WO2018101723A1 - Novel functionally enhanced mesenchymal progenitor cells, anti-inflammatory cell therapeutic agent composition containing same, and mesenchymal progenitor cell preparation method - Google Patents

Abstract

One embodiment of the present invention provides novel functionally enhanced mesenchymal progenitor cells overexpressing epidermal growth factor receptor (EGF-R) in comparison with mesenchymal stem cells (MSCs), and downexpressing cluster differentiation 95 (CD95) in comparison with the MSCs.

Description

Novel mesenchymal progenitor cells with improved functionality, anti-inflammatory cell therapeutic compositions comprising the same, and methods for producing mesenchymal progenitor cells

The present invention provides a novel functionally enhanced mesenchymal progenitor (FEMP) that overexpresses the epidermal growth factor receptor (EGF-R) and low expresses CD95 (Cluster Differentiation 95). It relates to an anti-inflammatory cell therapy composition comprising and a method for producing mesenchymal progenitor cells.

Mesenchyme refers to the tissues of mesoderm corresponding to the mesenchyme formed by Epithelial to Mesenchyme Transition (EMT), which occurs during embryonic development. Human embryonic stem cells can explain this early development in vitro. It can be used as the only source of beneficial cells.

Currently, there are active studies on mesenchymal stem cells or mesenchymal precursor cells derived from human embryonic stem cells as well as adults, suggesting that there are progenitor cells capable of forming bone in bone marrow like mesenchymal stem cells in adults. In addition to the bone marrow, it has been reported for the first time that it can be separated from dense bone, peripheral blood, adipose tissue, and fetal tissues such as cord blood and amniotic membrane.

Since adult-derived mesenchymal stem cells have various characteristics that can be useful for cell therapy, myocardial regeneration, pulmonary fibrosis, and spinal cord injury in the musculoskeletal system and cardiovascular system such as bone, cartilage and tendon in actual clinical treatment Attempts have been made in cell therapy in various fields.

However, in general, adult mesenchymal stem cells, unlike embryonic stem cells, are known to exhibit irregular proliferative capacity when they are passaged in vitro for a long time, so that the division capacity of one cell does not exceed 40 times. In addition, there is no established cell lineage, which requires repeated cell preparation from an adult, which limits the research.

In order to overcome these limitations, studies are being conducted to isolate and culture mesenchymal stem cells from human embryonic stem cells. A method of obtaining mesenchymal stem cells from known embryonic stem cells, which isolates cells displaying a desired marker by a flow activated analyzer (FACS) and cytokines to induce differentiation. The method of processing is generally used.

However, the method using a flow cytometer does not only lower the viability of the cells because of the use of a laser, but also increases the incubation period due to the small number of cells obtained after separation, and in the case of cytokine treatment, Even after differentiation, there is a problem that it is difficult to effectively remove the pluripotency of embryonic stem cells.

On the other hand, the use of such stem cells to progenitor cells is being actively researched for use as a cell therapy. Of these, no clear mechanism has been identified to date with regard to anti-inflammatory cell therapeutics, and no clear therapeutic effect appears.

The present invention is to solve the above-mentioned problems of the prior art, an object of the present invention is a novel mesenchymal progenitor cell having improved proliferative capacity and differentiation capacity and improved mobility, angiogenesis efficacy and body viability, cell therapeutic composition comprising the same And it provides a method for producing such mesenchymal progenitor cells.

However, the problem to be solved by the present invention is not limited to the above-mentioned problem, another task that is not mentioned will be clearly understood by those skilled in the art from the following description.

According to an embodiment of the present invention, overexpression of epidermal growth factor receptor (EGF-R, Epidermal Growth Factor Receptor) compared to mesenchymal stem cells (MSC, Mesenchymal Stem Cell), CD95 (Cluster Differentiation) compared to mesenchymal stem cells Functionally Enhanced Mesenchymal Progenitors (FEMPs) are provided that underexpress 95).

According to one side, the mesenchymal progenitor cells (FEMP) are embryonic stem cells (ESC, Embryonic Stem Cell), induced pluripotent stem cells (iPSC) or somatic cell nuclear transfer stem cells (SCNT, Somatic Cell Nuclear) Transfer cell).

According to one side, the embryonic stem cells may be derived from mammals.

According to one side, the mesenchymal progenitor cells (FEMP) is compared to mesenchymal stem cells Latent-Transforming Growth Factor Beta-binding Protein (LTBP), Vascular Endothelial Growth Factor (VEGF), Kinase insert Domain Receptor (KDR), FZD One or more angiogenesis factors selected from the group consisting of (Frizzled), Fibroblast Growth Factor (FGF), Angiopoietin (ANG), Platelet-Derived Growth Factor Receptor (PDGFR), and Endothelial Monocyte-Activating Polypeptide (EMAP). Can overexpress.

According to one side, the mesenchymal progenitor cells (FEMP) may overexpress one or more migration factors of Matrix Metalloproteinase (MMP) and Hepatocyte growth factor (HGF) compared to mesenchymal stem cells.

According to one side, the mesenchymal progenitor cells (FEMP) may have differentiation ability into adipocytes, osteocytes, osteoblasts, chondrocytes or myocytes.

According to one side, the mesenchymal progenitor cells (FEMP) may have a doubling time (20 hours to 35 hours).

According to one side, the mesenchymal progenitor cells (FEMP) can inhibit the proliferation of Peripheral Blood Mononuclear Cell (PBMC).

According to one side, the mesenchymal progenitor cells (FEMP) can induce the secretion of IL-10 (Interleukin 10).

According to another embodiment of the present invention, there is provided an anti-inflammatory cell therapy composition comprising the mesenchymal progenitor cells (FEMP) as an active ingredient.

According to another embodiment of the present invention, (a) the embryonic stem cells (ESC), induced pluripotent stem cells (iPSC) or somatic cell nuclear transfer stem cells (SCNT) derived from the embryonic body (Embryoid body) is provided with a porous membrane Culturing on the prepared cell culture insert; (b) separating progenitor cells that have migrated to the lower part of the cell culture insert; And (c) comparing epidermal growth factor receptor (EGF-R) and CD95 expression levels of the progenitor cells with the expression levels of mesenchymal stem cells (MSCs). do.

According to one side, the pore size of the porous membrane may be 1㎛ to 20㎛.

According to one side, the cell culture insert may comprise one or more additives selected from the group consisting of Fetal bovine serum (FBS), Serum replacement (SR) and Human platelet lysate (HPL).

Mesenchymal progenitor cells of the present invention may exhibit excellent proliferative, differentiating, migrating and angiogenic efficacy according to changes in specific marker expression patterns.

In addition, the cell therapeutic composition comprising such mesenchymal progenitor cells can implement an excellent anti-inflammatory effect and can be applied as an effective therapeutic agent for the site of inflammation caused by atopic dermatitis, cystitis and the like.

Furthermore, by producing the mesenchymal progenitor cells using a cell culture insert with a porous membrane, it is possible to improve cell viability and effectively remove the pluripotency of embryonic stem cells at low cost.

It is to be understood that the effects of the present invention are not limited to the above effects, and include all effects deduced from the configuration of the invention described in the detailed description or claims of the present invention.

1 is an image taken of mesenchymal progenitor cells (FEMPs) according to an embodiment of the present invention.

Figure 2 is an image of each step of the method for producing mesenchymal progenitor cells (FEMP) according to an embodiment of the present invention.

Figure 3 is a graph measuring the level of Oct4 and Nanog expression of mesenchymal progenitor cells (FEMP) according to an embodiment of the present invention.

Figure 4 is an image of the cell morphology according to the culture conditions of mesenchymal progenitor cells (FEMP) according to an embodiment of the present invention.

Figure 5 shows the results of karyotype analysis of mesenchymal progenitor cells (FEMP) according to an embodiment of the present invention.

Figure 6 is a graph measuring the EGF-R and CD95 expression rate of mesenchymal progenitor cells (FEMP) and bone marrow-derived mesenchymal stem cells (BM-MSC) according to an embodiment of the present invention.

Figure 7 is an image of the adipocytes and osteocytes (Osteocyte) differentiated mesenchymal progenitor cells (FEMP) according to an embodiment of the present invention.

Figure 8 shows the effect of inhibiting apoptosis by Fas Ligand of mesenchymal progenitor cells (FEMP) according to an embodiment of the present invention.

9 is an image and a graph measuring the movement characteristics of mesenchymal progenitor cells (FEMP) according to an embodiment of the present invention.

10 is a graph showing the expression level of angiogenesis factor and migration factor of mesenchymal progenitor cells (FEMP) according to an embodiment of the present invention compared with bone marrow-derived mesenchymal stem cells ( cutoff: 1.1).

11 is a graph showing PBMC proliferation inhibitory activity of mesenchymal progenitor cells (FEMP) according to an embodiment of the present invention.

12 is a graph showing IL-10 secretion inducing ability of mesenchymal progenitor cells (FEMP) according to an embodiment of the present invention.

13 is an image observing the efficacy of cystitis treatment of mesenchymal progenitor cells (FEMP) according to an embodiment of the present invention.

Hereinafter, exemplary embodiments will be described in detail with reference to the accompanying drawings.

Various modifications may be made to the embodiments described below. The examples described below are not intended to be limited to the embodiments and should be understood to include all modifications, equivalents, and substitutes for them.

The terminology used herein is for the purpose of describing particular example embodiments only and is not intended to be limiting of examples. Singular expressions include plural expressions unless the context clearly indicates otherwise. In this specification, terms such as "comprise" or "have" are intended to indicate that there is a feature, number, step, action, component, part, or combination thereof described on the specification, and one or more other features. It is to be understood that the present invention does not exclude the possibility of the presence or the addition of numbers, steps, operations, components, components, or a combination thereof.

Unless defined otherwise, all terms used herein, including technical or scientific terms, have the same meaning as commonly understood by one of ordinary skill in the art. Terms such as those defined in the commonly used dictionaries should be construed as having meanings consistent with the meanings in the context of the related art and shall not be construed in ideal or excessively formal meanings unless expressly defined in this application. Do not.

In addition, in describing the embodiment, when it is determined that the detailed description of the related known technology may unnecessarily obscure the gist of the embodiment, the detailed description thereof will be omitted.

Functionally Enhanced Mesenchymal Progenitors (FEMPs)

1 is an image taken of mesenchymal progenitor cells (FEMPs) according to an embodiment of the present invention.

According to one embodiment of the present invention as shown in Figure 1, overexpression of epidermal growth factor receptor (EGF-R, Epidermal Growth Factor Receptor) compared to mesenchymal stem cells (MSC, Mesenchymal Stem Cell), compared to mesenchymal stem cells Mesenchymal progenitor cells (FEMPs) are provided that underexpress CD95 (Cluster Differentiation 95).

"Mesenchymal stem cell (MSC)" refers to a multipotent stem cell (multipotent stem cell) capable of differentiating into a variety of mesodermal cells, including bone, cartilage, fat, muscle cells. The mesenchymal stem cells may be derived from umbilical cord, umbilical cord blood, bone marrow, fat, muscle, nerve, skin, amniotic membrane, chorion, decidual or placenta, preferably bone marrow-derived mesenchymal stem cells (BM-MSC, Bone Marrow-Mesenchymal Stem Cell).

In addition, the mesenchymal stem cells may be derived from mammals other than humans, fetuses or humans. Mammals other than humans include, but are not limited to, canines, felines, monkeys, cattle, sheep, pigs, horses, rats, mice, and guinea pigs.

The mesenchymal progenitor cells (FEMP) overexpress epidermal growth factor receptor (EGF-R) and low expression of CD95, compared to conventional mesenchymal stem cells (MSCs), and thus superior to the mesenchymal stem cells. It possesses proliferative capacity, differentiation capacity, and mobility, and can also exhibit excellent body viability. That is, the mesenchymal progenitor cells (FEMP) may refer to cells that are much more functional than the adult adult mesenchymal stem cells.

The epidermal growth factor receptor (EGF-R) is a cell-surface receptor for the epidermal growth factor family of extracellular protein ligands, which promotes paracrine of stem cells and results in VEGF (Vascular Endothelial Growth Factor). ), And may promote the expression of angiogenesis factors such as Fibroblast Growth Factor (HGF), and increase the expression of various growth factors or cytokines such as Heparin-Binding EGF-like Growth Factor (HBEGF) and Amphiregulin (AREG). Can improve stem cell proliferation, differentiation ability.

In addition, the EGF-R can act on stem cells with MAP kinase (Mitogen-Activated Protein kinase), PKC (Protein Kinase C) and the like to improve the mobility. Thus, mesenchymal progenitor cells (FEMP) overexpressing EGF-R may exhibit superior proliferative, differentiating and migrating ability compared to conventional mesenchymal stem cells.

Furthermore, in inflammatory diseases such as cystitis and atopic dermatitis, the EGF-R may promote anti-inflammatory activity and skin regeneration through interaction with epidermal growth factor (EGF).

The CD95 (Cluster Differentiation 95) is also known as FasR (Fas Receptor), APO-1 (Apoptosis antigen 1), or TNFRSF6 (Tumor Necrosis Factor Receptor Superfamily Member 6), and is located on the cell surface to apoptosis ( It is a death receptor that induces apoptosis.

Specifically, when a ligand known as FasL (Fas Ligand) or CD95L (CD95 Ligand) binds to CD95, apoptosis may occur by forming a death-inducing signaling complex (DISC).

That is, when stem cells or progenitor cells overexpress the CD95, signaling of apoptosis may be activated by the CD95, whereas stem cells or progenitor cells that relatively express the CD95 may be apoptotic signaling. This can be suppressed and can exhibit the excellent proliferation ability. Therefore, the mesenchymal progenitor cells (FEMP) can have excellent proliferative capacity compared to mesenchymal stem cells (MSC), in particular bone marrow-derived mesenchymal stem cells (BM-MSC). This property may mean that in stem cell grafts, it possesses excellent cell viability.

The mesenchymal progenitor cells (FEMP) are derived from embryonic stem cells (ESC, Embryonic Stem Cell), induced pluripotent stem cells (iPSC) or somatic cell nuclear transfer stem cells (SCNT). Can be.

As used herein, the term "embryonic stem cell (ESC)" refers to the extraction of inner cell mass (ICM, Inner Cell Mass) from blastocyst embryos just before mammalian fertilized egg implants into the mother's womb and cultured in vitro. As one, it refers to a pluripotency stem cell that can differentiate into all the cells of the animal. In a broad sense, this concept includes an embryoid body derived from embryonic stem cells, an induced pluripotent stem cell (iPSC) and a somatic cell nuclear transfer stem cell (SCNT). It can be understood as.

The embryonic stem cells include, but are not limited to, all embryonic stem cells derived from mammals including humans, primates, cattle, pigs, sheep, horses, dogs, mice, rats, and livestock. Stem cells.

As the human embryonic stem cells, for example, H9 (James A. Thomson et. Al. Embryonic Stem Cell Lines Derived from Human Blastocysts. Science 1998 Dec 4; 282 (5395): 1827) and the like may be used. Without limitation, human embryonic stem cells can be readily constructed by those skilled in the art.

Meanwhile, the mesenchymal progenitor cells (FEMP) are latent-transforming growth factor beta-binding protein (LTBP), vascular endothelial growth factor (VEGF), Kinase insert Domain Receptor (KDR), and FZD compared to mesenchymal stem cells (MSC). One or more angiogenesis factors selected from the group consisting of (Frizzled), Fibroblast Growth Factor (FGF), Angiopoietin (ANG), Platelet-Derived Growth Factor Receptor (PDGFR), and Endothelial Monocyte-Activating Polypeptide (EMAP). Can overexpress.

In addition, the mesenchymal progenitor cells (FEMP) may overexpress one or more migration factors of matrix metalloproteinase (MMP) and hepatocyte growth factor (HGF) compared to mesenchymal stem cells (MSC).

When the mesenchymal progenitor cells (FEMP) are present around the wounded tissue, anti-inflammatory activity, cell migration capacity and vascular regeneration efficacy may be important factors for the treatment thereof. That is, the stem cells may be moved to tissues or organs that are inflamed by a wound, thereby alleviating inflammation and sequentially regenerating damaged blood vessels. At this time, the effect of wound treatment may be determined according to the level of each anti-inflammatory activity, mobility and angiogenesis efficacy. The mobility may be understood as a concept including a homing ability to move to a target organ or tissue.

The mesenchymal progenitor cells (FEMPs) overexpress angiogenesis factors and migration factors compared to mesenchymal stem cells (MSCs), thereby rapidly moving to target organs or tissues to repair vascular tissues, thereby preventing or preventing inflammatory diseases. It can be usefully used as a therapeutic cell therapy.

In particular, FGF (Fibroblast Growth Factor) among the angiogenesis factors not only exhibits anti-inflammatory activity with the above-described epidermal growth factor receptor (EGF-R), but also promotes skin regeneration to treat inflammatory diseases such as atopic dermatitis. Can improve.

On the other hand, the mesenchymal progenitor cells (FEMP) as described above may have an excellent differentiation ability, for example, differentiation into adipocytes (adipocytes), osteocytes (osteocytes), chondrocytes (myocytes) or myocytes (myocytes).

Therefore, the mesenchymal progenitor cells can be used as a cell therapy suitable for the treatment of diseases, symptoms, such as diabetic retinopathy, interstitial cystitis, irritable cystitis, and may exhibit an excellent effect in the treatment of inflammatory diseases.

In addition, the mesenchymal progenitor cells (FEMP) can implement an enhanced proliferative capacity compared to conventional mesenchymal stem cells by overexpressing the epidermal growth factor receptor (EGF-R). Specifically, the doubling time of doubling the number of cells from the time of the stem cell culture (doubling time) may be 20 hours to 35 hours, preferably 24 hours to 32 hours. Such cell proliferation ability means that the effect of FEMP can be further improved as a cell therapy agent.

The mesenchymal progenitor cells (FEMP) may exhibit anti-inflammatory activity by inhibiting proliferation of Peripheral Blood Mononuclear Cells (PBMCs) or inducing secretion of Interleukin 10 (IL-10). Accordingly, the cell therapy composition may be applied as an effective therapeutic agent for atopic dermatitis, rheumatoid arthritis, interstitial cystitis and the like. The PBMC may include T-cells, B-cells, natural killer (NK) cells, monocytes, macrophages, and the like.

Specifically, the mesenchymal progenitor cells (FEMP) can inhibit the expression of Major Histocompatibility Complex (MHC) in macrophages and inhibit T-cell activity by inhibiting the interaction of CD28, CD80, CD86 and the like.

In addition, IL-10 secreted by mesenchymal progenitor cells (FEMP) inhibits the expression of inflammation-related enzymes such as inducible Nitric Oxide Synthase (iNOS) and Inducible Cyclooxyganas (COX-2) in macrophages. Can be represented. Specifically, the mesenchymal progenitor cells (FEMP) may increase the secretion of IL-10 in PBMC.

According to the excellent anti-inflammatory activity as described above, the mesenchymal progenitor cells can be used as an anti-inflammatory cell therapy composition.

In addition, the cell therapy composition may further include a support for receiving the mesenchymal progenitor cells (FEMP), preferably a biodegradable support. In this case, the biodegradable support may be a hydrogel such as fibrin glue, hyaluronic acid, gelatin, collagen, alginic acid, cellulose, pectin, chitin, polyglycolic acid, or polylactic acid, but is not limited thereto.

The cell therapy composition may further include a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier may be used in combination with saline, sterile water, Ringer's solution, buffered saline, dextrose solution, maltodextrin solution, glycerol, ethanol, HSA (Human serum albumin) and one or more of these components. If desired, other conventional additives such as antioxidants, buffers and bacteriostatic agents can be added.

The cell therapy composition may be formulated into an injectable formulation such as an aqueous solution, suspension, emulsion, or the like by additionally adding a diluent, a dispersant, a surfactant, a binder, and a lubricant, but is not limited thereto.

Method of manufacturing new mesenchymal progenitor cells (FEMP) with enhanced functionality

According to another embodiment of the present invention, (a) embryonic stem cells (ESC), induced pluripotent stem cells (iPSC) or somatic cell nuclear transfer stem cells (SCNT) derived embryonic body (Embryoid body) is provided with a porous membrane Culturing on a cell culture insert; (b) separating progenitor cells that have migrated to the lower part of the cell culture insert; And (c) comparing epidermal growth factor receptor (EGF-R) and CD95 expression levels of the progenitor cells with the expression levels of mesenchymal stem cells (MSCs). do.

Embryonic stem cells, induced pluripotent stem cells and somatic cell nuclear transfer stem cells of step (a) are as described above.

As used herein, the term “embryoid body (EB)” refers to a cell aggregate formed by attachment between embryonic stem cells, and when inoculated into a hydrophobic culture vessel, the embryonic stem cells are removed from the elements that maintain the undifferentiated state. Embryonic stem cells do not adhere to the bottom of the culture vessel and aggregate between cells to form embryoid bodies having a size of 1 mm or less. The embryoid body may have a trioderm (endoderm, mesoderm, ectoderm) differentiating ability constituting the body composition of the mammal.

As used herein, the term "cell culture insert" is a device having a porous membrane, epithelium that occurs during embryonic development when cultured embryonic stem cells, induced pluripotent stem cells or somatic cell nuclear transfer stem cells derived embryonic- Epithelial-Mesenchymal Transition (EMT) refers to the mechanism by which naturally occurs. At this time, the pore size of the porous membrane may be 1㎛ to 20㎛, preferably 6㎛ to 12㎛, more preferably 8㎛.

EGM2-MV, DMEM, MEM-α, STEMPRO-MSC, MesenCult-MSC medium can be used as the medium of the cell culture insert, preferably EGM2-MV, DMEM or MEM-α, It is not limited to this.

In addition, the cell culture insert may include one or more additives selected from the group consisting of Fetal bovine serum (FBS), Serum replacement (SR) and Human platelet lysate (HPL).

Specifically, the additive may be 1-10% FBS, 1-10% SR or 1-10% HPL, preferably 5% FBS or 2.5-5% HPL, but is not limited thereto. no.

In the step (b), after the step (a), the progenitor cells, specifically mesenchymal progenitor cells (FEMP), may be separated from the cell culture insert through the porous membrane. At this time, the FEMP may be aggregated in an epithelial cell shape under the cell culture insert.

Separation of the multifunctional mesenchymal progenitor cells (FEMP) may be performed by separating into single cells through enzymatic separation, or by separating into epithelial cell-like sheets through mechanical separation, for maintaining the prototype of stem cells. It may be desirable to use mechanical methods.

As used herein, the term "enzymatic separation" refers to a method for separating cell aggregates through enzymatic treatment, specifically collagenase (Collagense), accutase including collagenase I, II, III, IV (Accutase), Dispase, trypLE or trypsin-EDTA can be isolated by treatment, preferably may be a hypoallergenic enzyme trypLE, but is not limited thereto.

In addition, as a mechanical separation method, a method of separating and culturing in the state of maintaining the original shape of the epithelial cell sheet using a scraper can be used.

Step (c) compares the expression level of specific markers of progenitor cells isolated in step (b) with the expression levels of mesenchymal stem cells (MSC), specifically bone marrow-derived mesenchymal stem cells (BM-MSC). As such, the markers may be epidermal growth factor receptor (EGF-R) and CD95.

Accordingly, when the progenitor cells isolated in step (c) overexpress epidermal growth factor receptor (EGF-R) and low CD95 expression compared to the mesenchymal stem cells, multifunctional mesenchymal progenitor cells (FEMP) It can be judged as Specific marker characteristics are as described above.

Since the mesenchymal progenitor cells prepared according to the above method are particularly excellent in anti-inflammatory activity, they can be selected and formulated by mixing with a support, a carrier, a diluent, and other additives according to marker expression levels such as EGF-R and CD95. The support, carrier and the like are as described above.

Hereinafter, embodiments of the present invention will be described in detail.

Example 1 Isolation of Human Embryonic Stem Cells (hESC) -derived Mesenchymal Progenitor Cells (FEMP)

Figure 2 is an image of each step of the method for producing mesenchymal progenitor cells according to an embodiment of the present invention.

Referring to Figure 2, human embryonic stem cells (A) in the normal oxygen state (37 ℃, 5% CO ₂ cell incubator) after initial differentiation through the formation of embryoid body (B), cells having a pore size of 8 ㎛ Smear on culture inserts (C). Dulbecco's Modified Eagle Medium / Nutrient Mixture F-12 (DMEM / F12) containing 20% Serum Replacement (SR), 1% Non-essential amino acid (NEAA), 0.1% β-mercaptoethanol, and 1% anti-biotic Medium was used.

Embryos were cultured on the cell culture inserts for 1 to 10 days, thereby confirming that progenitor cells migrated below the inserts to form epithelial cell-like sheets (D). RT-PCR analysis confirmed the transition pattern of each cell and the results are shown in FIG. 3. Specific conditions such as marker gene, primer sequencing and the like are shown in Table 1 below.

Referring to FIG. 3, the human embryonic stem cells (hESC) remaining on the cell culture insert express a certain level of pluripotency markers Oct4 and Nanog, while the mesenchymal cells migrated to the cell culture insert below. Progenitor cells (FEMP) were found to express little Oct4 and Nanog similarly to human embryonic fibroblasts (hEF).

Through these results, mesenchymal progenitor cells (FEMPs) migrated to the lower cell culture inserts through epithelial-mesenchymal transition can be easily separated, and the mesenchymal progenitor cells (FEMPs) are human embryonic stem cells (hESCs). It can be expected to show a different characteristic from).

Example 2 Culture and Proliferation of Mesenchymal Progenitor Cells (FEMP) Derived from Human Embryonic Stem Cells (hESC)

Figure 2 is an image of each step of the method for producing a stem cell according to an embodiment of the present invention.

Referring to Figure 2, in Example 1, the mesenchymal progenitor cells (FEMP) sheet formed in the lower part of the cell culture was scraped off using a scraper (E) and then passaged to a new culture dish and cultured. At this time, the culture medium was EGM-2MV medium containing 5% fetal bovine serum (FBS, Fetal Bovine Serum).

Specifically, when stem cells extend from the epithelial cell-like sheet, the epithelial cell-shaped sheet is rolled out, and passaged again to obtain stem cells continuously. Remaining stem cells were removed and passaged using trypLE.

As a result of observing the progenitor cells of the 1st passage (F), 5th passage (G), and 7th passage (H), no abnormalities were found in the shape and characteristics of the progenitor cells even after repeated passages of several passages. These results indicate that the mesenchymal progenitor cells (FEMP) derived from human embryonic stem cells have no restriction in passage, and a method of using a porous membrane to separate and culture human embryonic stem cell-derived mesenchymal stem cells has been previously reported. It is efficient compared to.

In addition, subcultured mesenchymal progenitor cells (FEMP) under the same conditions as above, but using 2.5% and 5% of HPL (Human platelet lysate) replacing 5% of FBS, stem cells as in the case of using FBS It could be confirmed that it extends (FIG. 4).

Example 3: Chromosome Analysis of Mesenchymal Progenitor Cells (FEMP)

In order to confirm whether mesenchymal progenitor cells (FEMP) prepared according to Examples 1 and 2 exhibited normal chromosomal characteristics regardless of the number and duration of passages, karyotyping of 9 passages and 19 passages was performed.

Specifically, 20 μl of a colchicine solution having a concentration of 100 μg / μl in a culture vessel containing 10 ml of the culture solution was mixed and left at 37 ° C. for 2 hours. Thereafter, the solution was centrifuged at 500 rpm, the supernatant was removed and the cells were suspended in 5 ml stock solution (0.075 M KCl) and left in a constant temperature water bath at 37 ° C. for 25 minutes. 5 drops of fixative solution (methanol: glacial acetic acid = 3: 1, v / v) was added to each tube, centrifuged at 1,200 rpm for 8 minutes, the supernatant was removed, and the cells were suspended in 5 ml fixative solution. The procedure of leaving for 10 minutes was repeated twice. The cells were suspended in 0.5 ml of fixed solution and cell suspension was added dropwise onto a slide glass immersed in 70% ethanol. Ethanol on the slide was dried using an alcohol lamp, and water was removed from the air, and then the coverslip was covered and observed under a microscope (FIG. 5).

Referring to FIG. 5, it was confirmed that mesenchymal progenitor cells (FEMP) exhibited normal chromosomal characteristics regardless of the number and duration of passages.

Example 4: Marker Expression Analysis of Mesenchymal Progenitor Cells (FEMP)

In order to confirm the cellular characteristics of 11 passage and 21 passage mesenchymal progenitor cells (FEMP) prepared according to Example 2, the expression of the marker was analyzed using a flow cytometer (FACS, Fluorescence Activated Cell Sorting). Specifically, stem cells were detached using trypsin-EDTA, and then suspended in PBS (Phosphate Buffered Saline) at a concentration of ⁵ × 10 ⁵ cells / ml. CD73, CD95, CD44, CD11b, CD14, CD19, CD34, CD45, Human Leukocyte Antigen-Antigen D Related (HLA-DR) and Epidermal Growth Factor Receptor (EGF-R) antibodies were added to each cell for 45 minutes at room temperature. After the reaction, flow cytometry was performed on each cell, and the results are shown in Table 2 below, and the results of EGF-R and CD95 were shown together in FIG. 6. At this time, the control group used bone marrow-derived mesenchymal stem cells (BM-MSC) of the seventh generation and fibroblasts of the 16th generation (Fibroblast).

Referring to Table 2 and Figure 6, mesenchymal progenitor cells (FEMP) compared to BM-MSC overexpressing EGF-R about 40% to 70%, it can be seen that CD95 is about 80% low expression. Through these results, it can be expected that mesenchymal progenitor cells (FEMP) show superior proliferation and differentiation capacity compared to BM-MSC.

In addition, markers such as CD73 were strongly expressed, and hematopoietic stem cell markers such as CD34 and CD45 were not expressed, and thus, the mesenchymal progenitor cells (FEMP) were analyzed to include the characteristics and multipotency of conventional mesenchymal stem cells. In fact, it was confirmed that the differentiation into adipocytes, bone cells, myocytes and the like (FIG. 7).

Example 5 Analysis of Apoptosis in Mesenchymal Progenitor Cells (FEMP) and BM-MSCs by Fas Ligand

To confirm Fas Ligand-induced apoptosis, mesenchymal progenitor cells were treated with recombinant Fas Ligand. At this time, bone marrow-derived mesenchymal stem cells (BM-MSC) were used as a control, and A549 was used as a positive control group. Specifically, seeding of BM-MSC, mesenchymal progenitor cells (FEMP), and A549 cells at 1 × 10 ⁴ cells / well at 100 μl was performed on each 96 well culture plates, and cultured for 37 to 24 hours, followed by apoptosis. In order to induce recombinant human Fas Ligand was reacted for 24 hours at a concentration of 0, 50, 100, 500 ng / ㎖. To identify the population of apoptotic cells, cell viability was analyzed by CCK-8 reaction. The analysis results are shown in FIG. 8.

Referring to FIG. 8, apoptotic cells increased in a concentration-dependent manner when Fas Ligand was treated in BM-MSC. In particular, it was confirmed that apoptotic cells increased by 40.64 ± 12.02% on day 1 after treatment with 500ng / ml Fas Ligand. However, mesenchymal progenitor cells (FEMP) were found to increase 21.19 ± 7.21%.

These results confirmed that the mesenchymal progenitor cells (FEMP) are CD95 low-expressing cells, so that apoptosis by Fas Ligand is less observed, and BM-MSCs are CD95 high-expressing cells, thereby promoting apoptosis. This result influences the growth and apoptosis of MSC depending on Fas Ligand depending on CD95 expression level.

Example 6: Characterization of proliferation of mesenchymal progenitor cells (FEMP)

In order to confirm the proliferative characteristics of mesenchymal progenitor cells (FEMP) prepared according to Examples 1 and 2, the cell proliferation ability of 16 passage mesenchymal progenitor cells (FEMP) cultured under normal oxygen partial pressure was measured in viable cells. Analysis was performed using density (cells / cm ² ). At this time, 16 passages bone marrow-derived mesenchymal stem cells (BM-MSC) cultured under normal oxygen partial pressure were used as a control.

Each stem cell was plated with 3 wells of each passage (16, 17, 18, 19 passages) at a concentration of 2.0x10 ⁴ cells / plate in a 6 well plate dish. Each stem cell was cultured at normal oxygen partial pressure, and then separated from the culture dish using 0.25% trypsin. The average cell number was confirmed by counting the cell number three times using a hemocytometer, and the results are shown in Table 3 below.

Referring to Table 3, the mesenchymal progenitor cells (FEMP) prepared according to Examples 1 and 2 can be seen that the doubling time is significantly reduced compared to the bone marrow-derived mesenchymal stem cells (BM-MSC) have.

Specifically, while BM-MSCs showed a doubling time of more than 39 hours, mesenchymal progenitor cells (FEMPs) showed a doubling time of less than 32 hours, indicating a significantly improved proliferative capacity.

Example 7: Analysis of migration characteristics of mesenchymal progenitor cells (FEMP)

To determine the migration characteristics of the mesenchymal progenitor cells (FEMP) prepared according to Examples 1 and 2 above, the number of migrating cells was measured using μ-dish 35 mm and high culture-insert. Analyzed. At this time, bone marrow-derived mesenchymal stem cells (BM-MSC) was used as a control.

Specifically, the mesenchymal progenitor cells (FEMP) and BM-MSC were seeded at 70 μl each at 3 × 10 ⁴ cells / well at 35 μm of each dish, and incubated at 37 ° C. for 24 hours. Culture-inserts were removed, 2 ml of 10% DMEM was added, photographed under a microscope and the number of mobile cells counted. The analysis results are shown in FIG. 9.

Referring to Figure 9, it can be seen that the number of mobile cells of mesenchymal progenitor cells (FEMP) compared to BM-MSC is much higher, these results show that the mesenchymal progenitor cells (FEMP) has excellent anti-inflammatory activity through excellent mobility Imply that it can be shown.

Example 8 Analysis of Angiogenic and Molecular Factors of Mesenchymal Progenitor Cells (FEMP)

In order to confirm the expression characteristics of angiogenesis factor and migration factor of mesenchymal progenitor cells (FEMP) of Examples 1 and 2, 11 mesenchymal cells in the same manner as in Example 4 The expression rate of progenitor cells (FEMP) and bone marrow-derived mesenchymal stem cells (BM-MSC) of seven passages was analyzed.

The results of the analysis are expressed as a fold change of FEMP / BM-MSC, and the cutoff value is set to 1.1 and is shown in FIG. 10.

Referring to Figure 10, the mobile factors MMP-1 and HGF is expressed in mesenchymal progenitor cells (FEMP) more than 1.3 times compared to BM-MSC is analyzed that the mesenchymal progenitor cells (FEMP) shows excellent mobility , It can be seen that this is the result consistent with Example 7.

In addition, compared with BM-MSC, various angiogenic factors such as LTBP-1, ANG-1, and FGF-16 are expressed at high levels in mesenchymal progenitor cells (FEMP), particularly LTBP-1, VEGF-B, KDR, In the case of Frizzled-1 and FGF-16, 1.3-fold or more expression of the mesenchymal progenitor cells (FEMP) is compared with BM-MSC.

Through these results, it can be seen that the mesenchymal progenitor cells (FEMP) can implement excellent efficacy as a cell therapy for angiogenesis, ie damage vessel regeneration as well as anti-inflammatory as described below.

Example 9: Analysis of PBMC proliferation inhibitory effect of mesenchymal progenitor cells (FEMP)

In order to confirm the effect of inhibiting Peripheral Blood Mononuclear Cell (PBMC) proliferation of mesenchymal progenitor cells (FEMP) prepared according to Examples 1 and 2, by varying the number of cells (n = 1000, 2000, 5000, 10000) Mesenchymal progenitor cells (FEMP) and bone marrow-derived mesenchymal stem cells (BM-MSC) were co-cultured for 5 days in 96 well plates containing Peripheral Blood Mononuclear Cells (PBMC) at a concentration of 2 × 10 ⁵ cells / well. For each experimental group was labeled with CFSE (CarboxyFluorescein Succinimidyl Ester) to measure the inhibition rate of PBMC growth (%), the results are shown in Table 4 and FIG.

Referring to Table 4 and FIG. 11, the mesenchymal progenitor cells (FEMP) showed improved PBMC inhibition rate compared to BM-MSC in the same cell number, and in particular, in the experimental group having 10,000 cell numbers, the inhibition rate was approximately 10-fold. .

The results suggest that mesenchymal progenitor cells (FEMP) prepared according to Examples 1 and 2 can be applied as a cell therapy showing excellent anti-inflammatory activity by inhibiting the proliferation of PBMCs formed by blood vessels, tissue damage and the like.

Experimental Example 10 Analysis of IL-10 Secretion Induction Effect of Mesenchymal Progenitor Cells (FEMP)

In order to confirm the anti-inflammatory activity of the mesenchymal progenitor cells (FEMP) prepared according to Examples 1 and 2, specifically IL-10 (Interleukin 10) secretion inducing effect, Peripheral Blood Mononuclear at a concentration of 1x10 ⁶ cells / well Cells) were plated in a 96 well plate and induced an inflammatory response at 100 ng / ml LPS.

Specifically, the concentration of IL-10 in each experimental group was measured with or without LPS treatment and cells (FEMP or BM-MSC) treatment, and the results are shown in Table 5 and FIG. 12.

Referring to Table 5 and FIG. 12, it can be seen that a certain level of IL-10 is secreted from PBMCs that induce an inflammatory response with LPS compared to before adding LPS. In addition, IL-10 was further increased when PBMCs, which caused inflammatory responses with LPS, were co-cultured with BM-MSC or FEMP.

These results indicate that not only bone marrow-derived mesenchymal stem cells (BM-MSCs), but also mesenchymal progenitor cells (FEMPs) prepared according to Examples 1 and 2 also induce secretion of IL-10 by PBMCs in an inflammatory environment. Suggests anti-inflammatory activity. Therefore, the composition containing the FEMP can be applied as a therapeutic agent for anti-inflammatory.

Experimental Example 11: Analysis of cystitis treatment effect of mesenchymal progenitor cells (FEMP)

Animal models were prepared to confirm the therapeutic effect of cystitis of mesenchymal progenitor cells (FEMP) prepared according to Examples 1 and 2. A 24-gage catheter was inserted into the bladder of 6-week-old female SD rats to expose 0.2 mM HCl for 10 minutes, neutralized by treating with 1 M bicarbonate for 10 minutes, and washed twice with PBS to induce cystitis.

After one week, the abdominal cavity was opened to expose the bladder, and the mesenchymal progenitor cells were treated by concentration between the bladder outer layer and the inner membrane, and the control group was treated with PBS. Tissues were prepared by extracting the tissues at 4 and 8 weeks after cell transplantation, and H & E staining was performed.

Referring to FIG. 13, in the control group treated with PBS, infiltration of inflammatory cells and formation of granulation tissue were observed in the bladder lining over time, and hyperproliferation of epithelial cells was observed.

In the group treated with mesenchymal progenitor cells (FEMP) at low concentrations (1x10 ⁵ cell / 100µl, 1x10 ⁶ cell / 100µl), the results showed similar results to those of the control group, but no clear cystitis treatment effect was observed, but the concentration of 3x10 ⁶ or higher was treated. In one group, bladder epithelial and lamina propria tissues recovered to a similar extent as normal tissues.

From these results, it can be seen that the mesenchymal progenitor cells (FEMP) can be applied for the treatment of cystitis, in particular for the treatment of interstitial cystitis.

Although the embodiments have been described by the limited embodiments and the drawings as described above, various modifications and variations are possible to those skilled in the art from the above description. For example, the techniques described may be performed in a different order than the described method, and / or the components described may be combined or combined in a different form than the described method, or replaced or substituted by other components or equivalents. Appropriate results can be achieved.

Therefore, other implementations, other embodiments, and equivalents to the claims are within the scope of the following claims.

Claims (13)

Overexpress epidermal growth factor receptor (EGF-R) compared to mesenchymal stem cells (MSC); Mesenchymal progenitor cells that express CD95 (Cluster Differentiation 95) lower than mesenchymal stem cells. The method of claim 1, Mesenchymal progenitor cells derived from embryonic stem cells (ESC), induced pluripotent stem cells (iPSCs) or somatic cell nuclear transfer stem cells (SCNTs). The method of claim 2, The embryonic stem cells are mammalian origin, mesenchymal progenitor cells. The method of claim 1, Compared with mesenchymal stem cells, Latent-Transforming Growth Factor Beta-binding Protein (LTBP), Vascular Endothelial Growth Factor (VEGF), Kinase insert Domain Receptor (KDR), Frizzled (FZD), Fibroblast Growth Factor (FGF), and Angiopoietin (ANG) ), Mesenchymal progenitor cells overexpressing one or more angiogenesis factors selected from the group consisting of Platelet-Derived Growth Factor Receptor (PDGFR) and Endothelial Monocyte-Activating Polypeptide (EMAP). The method of claim 1, Mesenchymal progenitor cells that overexpress one or more migration factors of matrix metalloproteinase (MMP) and hepatocyte growth factor (HGF) compared to mesenchymal stem cells. The method of claim 1, Mesenchymal progenitor cells having differentiation capacity into adipocytes, osteocytes, chondrocytes or myocytes. The method of claim 1, Mesenchymal progenitor cells having a doubling time of 20 to 35 hours. The method of claim 1, Mesenchymal progenitor cells that inhibit the proliferation of Peripheral Blood Mononuclear Cells (PBMCs). The method of claim 1, Mesenchymal progenitor cells that induce the secretion of IL-10 (Interleukin 10). 10. The anti-inflammatory cell therapy composition comprising the mesenchymal progenitor cell of any one of claims 1 to 9 as an active ingredient. (a) culturing embryonic bodies derived from embryonic stem cells (ESC), induced pluripotent stem cells (iPSC) or somatic cell nuclear transfer stem cells (SCNT) on a cell culture insert with porous membrane; (b) separating progenitor cells that have migrated to the lower part of the cell culture insert; And (c) comparing the epidermal growth factor receptor (EGF-R) and CD95 expression levels of the progenitor cells with the expression levels of mesenchymal stem cells (MSCs). The method of claim 11, The pore size of the porous membrane is 1㎛ to 20㎛, method for producing mesenchymal progenitor cells. The method of claim 11, The cell culture insert comprises at least one additive selected from the group consisting of FBS (Fetal bovine serum), SR (Serum replacement) and HPL (Human platelet lysate), method for producing mesenchymal progenitor cells.

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