Musculoskeletal stem cell

Abstract

The present disclosure relates to a novel musculoskeletal stem cell (MSSC) differentiated from an ESC (embryonic stem cell) or an iPSC (induced pluripotent stem cell). The musculoskeletal stem cell of the present disclosure can be easily induced from a human embryonic stem cell or a human-derived pluripotent stem cell and can be effectively differentiated not only into bone but also into cartilage, tendon and muscle. Accordingly, it can be usefully used for prevention or treatment of various musculoskeletal diseases.

Claims

1. A musculoskeletal stem cell (MSSC) differentiated from an ESC (embryonic stem cell) or an iPSC (induced pluripotent stem cell), wherein the musculoskeletal stem cell has the following characteristics: a) positive for the ectodermal marker nestin (NES); b) positive for the myogenic satellite marker Pax7; c) positive for the mesodermal marker α-SMA; d) negative for the pluripotency marker LIN28; and f) negative for the mesenchymal stem cell marker CD90 wherein the musculoskeletal stem cell can be differentiated into bone, cartilage, tendon, ligament, muscle and fat.

2. The musculoskeletal stem cell according to claim 1, wherein the musculoskeletal stem cell is deposited in the Korean Cell Line Bank under the accession number KCLRF-BP-00460.

3. A pharmaceutical composition for preventing or treating a musculoskeletal disease, comprising the musculoskeletal stem cell according to claim 1.

4. The pharmaceutical composition according to claim 3, wherein the musculoskeletal disease is one or more disease selected from a group consisting of osteoporosis, osteomalacia, osteogenesis imperfecta, osteopetrosis, osteosclerosis, Paget's disease, bone cancer, arthritis, rickets, fracture, periodontal disease, segmental bone defect, osteolytic bone disease, primary and secondary hyperparathyroidism, hyperostosis, degenerative arthritis, degenerative knee joint disease, degenerative hip joint disease, degenerative foot joint disease, degenerative hand joint disease, degenerative shoulder joint disease, degenerative elbow joint disease, chondromalacia patellae, simple knee arthritis, osteochondritis dissecans, lateral epicondylitis, medial epicondylitis, Heberden's nodes, Bouchard's nodes, degenerative thumb CM arthrosis, meniscal injury, degenerative disc disease, cruciate ligament injury, biceps brachii muscle injury, ligament injury, tendon injury, frozen shoulder, rotator cuff tear, calcific tendinitis, shoulder impingement syndrome, recurrent dislocation, habitual dislocation, senile sarcopenia and muscular dystrophy.

Description

BRIEF DESCRIPTION OF DRAWINGS

(1) FIGS. 1A-1E show the characteristics of hMSSC differentiated from hESC. FIG. 1A shows the change in cell morphology of hESC subcultured with a medium for inducing differentiation into a musculoskeletal stem cell, from passage 7 to passage 19. FIG. 1B shows a result of observing the expression of the pluripotency markers OCT4, NANOG, SOX2 and LIN28 in hMSSC by immunocytochemistry. FIG. 1C shows a result of confirming the expression of pluripotency, ectodermal, mesodermal and endodermal markers in hESC, hMSC and hMSSC at passages 7 and 17 by RNA sequencing, twice respectively. FIG. 1D shows a result of measuring the expression of cell surface antigens by flow cytometry for characterization of hMSSC. FIG. 1E shows a result of analyzing the expression of various cell-specific markers by immunocytochemistry for characterization of hMSSC. In the figure, DAPI represents stained nuclei and the blue triangle indicates a β-galactosidase positive cell.

(2) FIGS. 2A-2E show a result of comparing the in-vitro differentiation capacity of hMSSC with other types of cells. FIG. 2A shows a result of comparing the in-vitro bone, cartilage and fat differentiation capacity of hMSC and hMSSC. FIG. 2B shows a result of confirming that hMSSC has the potential to differentiate into skeletal muscle by immunocytochemistry for the skeletal muscle cell-specific marker MYH9. C2C12 was used as a positive control group for the skeletal muscle cell. FIGS. 2C and 2D show a result of confirming that hMSSC lacks the potential to differentiate into an endothelial cell by immunocytochemistry for the endothelial cell-specific markers CD31 and VE-cadherin. FIG. 2E shows a result of confirming that hMSSC lacks the potential to differentiate into a nerve cell by immunocytochemistry for the nerve cell-specific marker MAP2. As a positive control group, neural stem cells differentiated from H9 hESC were used.

(3) FIGS. 3A-3C show a result of measuring the differentiability of hMSSC in vivo. FIG. 3A-a shows that muscle, fat and tendon were formed when hMSSC was transplanted into the kidney as confirmed by H&E staining. FIG. 3A-b shows a result of confirming the differentiation of hMSSC transplanted into the kidney into muscle, fat and tendon cells by immunohistochemistry for the muscle-specific marker pMLC, the fat-specific marker PPARgamma (PPAr) and ligament-specific marker Scx. hLA is a human cell-specific marker. The staining result shows that the cell is derived from human. FIG. 3B-a shows a result of micro-CT scanning showing that bone was formed at the site where hMSSC was transplanted into the kidney. FIGS. 3B-b and 3B-c show a result of confirming bone formation by H&E and pentachrome immunohistochemical staining. FIG. 3B-d shows a result of confirming the expression of the human cell marker hLA (human leukocyte antigen), the bone markers Osx (osterix), Runx2, DMP1 and OCN (osteocalin) and the vascular marker vWF in the cells of the osteoblastic tissue by immunohistochemistry. FIG. 3C shows a result of confirming that cartilage was formed as a result of differentiation of hMSSC transplanted into the hypoderm into cartilage cells by H&E and toluidine blue immunohistochemical staining. The expression of the cartilage marker ColII (collagen II) was also confirmed by immunohistochemistry.

(4) FIGS. 4A-4B show a result of confirming the effect of hMSSC on recovery of fracture. FIG. 4A shows that, when hMSC was transplanted into the fracture site, bone was formed by the cells of mouse itself, not by the hMSC. FIG. 4A-a shows micro-CT images obtained 2, 4 and 6 weeks after the transplantation of hMSC into the fracture site. FIG. 4A-b shows a result of H&E immunohistochemistry of the thighbone containing the fracture site into which hMSC was transplanted. FIG. 4A-c shows a result of magnifying the red square portion of 4A-b. FIG. 4A-d shows a result of confirming that the transplanted hMSC was not differentiated into bone cells by immunohistochemistry for the bone cell marker Runx2 and the human cell marker hLA. FIG. 4B shows that, unlike hMSC, bone was formed as transplanted hMSSC was differentiated. FIG. 4B-a shows micro-CT images obtained 2, 4 and 6 weeks after the transplantation of hMSSC into the fracture site. FIG. 4B-b shows a result of H&E immunohistochemistry of the thighbone containing the fracture site into which hMSSC was transplanted. FIG. 4B-c shows a result of magnifying the red square portion of 4B-b. FIG. 4B-d shows a result of confirming that the transplanted hMSSC was differentiated into bone cells by immunohistochemistry for the bone cell marker Runx2 and the human cell marker hLA.

(5) FIGS. 5A-5D show a result of investigating whether hiPSC is also differentiated into hMSSC like hESC. FIG. 5A shows a result of confirming the expression level of the pluripotency markers Oct4, Nanog, Sox2 and Lin28 in hMSSC differentiated from hiPS by immunocytochemistry. FIG. 5B shows a result of investigating the expression of specific cell surface antigens in hMSSC differentiated from hiPS by flow cytometry. FIG. 5C shows a result of confirming the in-vitro bone, cartilage and fat differentiation capacity of hMSSC differentiated from hiPS. FIG. 5D shows a result of differentiating hMSSC differentiated from hiPS into skeletal muscles in a medium for inducing differentiation into skeletal muscle and then conducting immunocytochemistry for the skeletal muscle marker MYH9.

(6) FIG. 6 shows a result of comparing the expression level of CD44 in a CM medium and in hMSSC differentiated using a medium for inducing differentiation into hMSSC by flow cytometry.

(7) FIG. 7 shows a result of Alcian blue staining for confirming differentiation into cartilage and ALP and Alizarin red S staining for confirming differentiation into bone in order to investigate the tendency to differentiation into cartilage or bone when some ingredients are absent in a medium for inducing differentiation into hMSSC.

MODE FOR INVENTION

(8) Hereinafter, the present disclosure will be described in detail through examples. However, the following examples are for illustrative purposes only and it will be apparent to those of ordinary skill in the art that the scope of the present disclosure is not limited by the examples.

EXAMPLES

(9) Experimental Materials and Methods

Example 1

Experimental Animals

(10) 7- to 10-week-old Balb/c-nude background mice (weighing 20-24 g) were purchased from Orient Bio (Seongnam, Korea). All animal experiments were performed according to the guidelines of the Chonbuk University Animal Care and Use Committee. The animals were accommodated under controlled-temperature (21-24° C.) and 12:12-hr light-darky cycle environments and were given free access to water and feed.

Example 2.1

Induction of Differentiation from hESC into hMSSC

(11) H9 hESCs (human embryonic stem cells) were purchased from WiCell (Madison, Mich., USA). The hESCs were cultured on CF1 mouse embryonic fibroblast (MEF) feeder cells whose cell division was blocked by mitomycin C treatment. A hESC culture medium was prepared as DMEM/F12 (Invitrogen, USA) supplemented with 20% knockout serum replacement (KSR; Invitrogen, USA), 1 mM glutamine (Invitrogen, USA), 1% nonessential amino acids (Invitrogen, USA), 0.1 mM β-mercaptoethanol (Invitrogen, USA), 0.1% penicillin/streptomycin (Invitrogen, USA) and 15 ng/mL bFGF (R&D Systems, USA).

(12) The hESCs were induced to differentiate into hMSSCs (human musculoskeletal stem cells) using a medium for inducing differentiation into MSSC (hereinafter, referred to as “MSSC medium”) of the following composition:

(13) 1) 250 ng/mL human noggin (KOMA Biotech, Korea),

(14) 2) 20 ng/mL human LIF (KOMA Biotech, Korea),

(15) 3) 15 ng/mL basic fibroblast growth factor (FGF) (R&D Systems, USA) (FGF2 signaling activator),

(16) 4) 3 μM CHIR99021 (Cayman, USA) (Wnt signaling activator),

(17) 5) 1 μM PD0325901 (Cayman, USA) (ERK (extracellular signal-regulated kinase) signaling inhibitor),

(18) 6) 10 μM SB431542 (Tocris, United Kingdom) (TGF-β/activin/nodal signaling inhibitor) and

(19) 7) 10% knockout serum replacement (Invitrogen, USA), 1% N2 supplement (Gibco, USA), 2% B27 supplement (Gibco, USA), 1% nonessential amino acids (Gibco, USA), 43% DMEM/F12 (Gibco, USA), 43% Neurobasal (Gibco, USA), 1 mM glutamine, 0.1 mM β-mercaptoethanol, 0.1% penicillin-streptomycin and 5 mg/mL bovine serum albumin (Gibco, USA).

(20) After treating the hESCs with ROCK (Rho-associated coiled-coil kinase) inhibitor (Y-27632, 10 μM, Calbiochem, Germany) and PKC (protein kinase C) inhibitor (Go6983, 2.5 μM, Sigma, USA) for 24 hours in order to enhance survivability and trypsinizing the hESCs by treating with TrypLE (Life Technologies, USA), they were induced to differentiate into hMSSCs by culturing with the MSSC medium on a culture dish coated with vitronectin and gelatin (1 ng/mL, Sigma, USA) until passage 7. The differentiated MSSC cells were identified to be stably identical from passage 5 and the cells cultured for 10 passages were deposited in the Korean Cell Line Bank on Oct. 10, 2018 and were given the accession number KCLRF-BP-00460.

Example 2.2

Induction of Differentiation from hiPSC into hMSSC

(21) hiPSCs (human induced pluripotent stem cells) were obtained by introducing the OCT4, KLF4, SOX2 and cMYC genes to BJ fibroblasts (ATCC®CRL2522™) using Sendai virus according to the method developed by Hasegawa et al. (Fusaki et al., 2009, PNAS 85, 348-362). The hiPSCs were cultured on CF1 mouse embryonic fibroblast (MEF) feeder cells whose cell division was blocked by mitomycin C treatment. A hiPSC culture medium was prepared as DMEM/F12 (Invitrogen, USA) supplemented with 20% knockout serum replacement (KSR; Invitrogen, USA), 1 mM glutamine (Invitrogen, USA), 1% nonessential amino acids (Invitrogen, USA), 0.1 mM β-mercaptoethanol (Invitrogen, USA), 0.1% penicillin/streptomycin (Invitrogen, USA) and 15 ng/mL bFGF (R&D Systems, USA).

(22) The hESCs were induced to differentiate into hMSSCs (human musculoskeletal stem cells) using a medium for inducing differentiation into MSSC (hereinafter, referred to as “MSSC medium”) of the following composition:

(23) 1) 250 ng/mL human noggin (KOMA Biotech, Korea),

(24) 2) 20 ng/mL human LIF (KOMA Biotech, Korea),

(25) 3) 15 ng/mL basic fibroblast growth factor (FGF) (R&D Systems, USA) (FGF2 signaling activator),

(26) 4) 3 μM CHIR99021 (Cayman, USA) (Wnt signaling activator),

(27) 5) 1 μM PD0325901 (Cayman, USA) (ERK (extracellular signal-regulated kinase) signaling inhibitor),

(28) 6) 10 μM SB431542 (Tocris, United Kingdom) (TGF-β/activin/nodal signaling inhibitor) and

(29) 7) 10% knockout serum replacement (Invitrogen, USA), 1% N2 supplement (Gibco, USA), 2% B27 supplement (Gibco, USA), 1% nonessential amino acids (Gibco, USA), 43% DMEM/F12 (Gibco, USA), 43% Neurobasal (Gibco, USA), 1 mM glutamine, 0.1 mM β-mercaptoethanol, 0.1% penicillin-streptomycin and 5 mg/mL bovine serum albumin (Gibco, USA).

(30) After treating the hiPSCs with ROCK (Rho-associated coiled-coil kinase) inhibitor (Y-27632, 10 μM, Calbiochem, Germany) and PKC (protein kinase C) inhibitor (Go6983, 2.5 μM, Sigma, USA) for 24 hours in order to enhance survivability and trypsinizing the hiPSCs by treating with TrypLE (Life Technologies, USA), they were induced to differentiate into hMSSCs by culturing with the MSSC medium on a culture dish coated with vitronectin and gelatin (1 ng/mL, Sigma, USA) until passage 7. The differentiated MSSC cells were identified to be stably identical from passage 5.

Example 3

Immunohistochemistry

(31) Samples obtained by injecting the hMSSCs differentiated in Example 2.1 into the hypoderm and kidney of Balb/c-nude as described in Examples 10.1 and 10.2 were fixed overnight at 4° C. in 2% paraformaldehyde (PFA; Wako, Japan). For a sample to investigate differentiation into bone, decalcification was conducted at 4° C. for 2 weeks in PBS (pH 7.2) using 0.4 M EDTA. Then, the samples were dehydrated using ethanol and xylene sequentially, embedded in paraffin and cut to 5 μm thickness. The cut surface was stained with H&E and modified Movat's pentachrome (Cosmobio, Japan).

Example 4

RNA Sequencing

(32) RNAs were extracted from H9 hESCs, human mesenchymal stem cells (hMSCs; Lonza, Switzerland) and the hMSSCs of Example 2.1 using Trizol reagent (Invitrogen, USA). The RNA quality was evaluated with the Agilent 2100 bioanalyzer and the RNA 6000 Nano Chip (Agilent Technologies, USA) and quantification was performed using the ND-2000 spectrophotometer (Thermo Inc., USA). An RNA library for RNA sequencing was established using the SENSE 3′ mRNA-Seq Library Prep Kit (Lexogen Inc., Australia). RNA sequencing was conducted using NextSeq 500 (Illumina Inc., USA). The SENSE 3′ mRNA-Seq reads were aligned using Bowtie2 version 2.1.0. The difference in gene expression was determined using Bioconductor R version 3.2.2 with EdgeR. The read count data were processed with Genowiz version 4.0.5.6 (Ocium Biosolutions, USA).

Example 5

Immunochemistry

(33) “Immunocytochemistry” was performed according to the following method.

(34) For immunofluorescence staining, the cells were fixed in 4% paraformaldehyde, made permeable with 0.5% Triton X-100 and then blocked with 10% normal goat, normal rabbit or fetal bovine serum in phosphate-buffered saline (PBS). The sample was stained overnight at 4° C. with primary antibodies against Tuj1 (Covance, USA), α-smooth muscle (α-SMA, Sigma, USA), Nanog (Santa Cruz, USA), Oct3/4 (Santa Cruz, USA), Sox2 (Santa Cruz, USA), CD31 (DAKO, Japan), vascular endothelial-cadherin (R&D, USA), MYH9 (Santa Cruz, USA), HNK-1 (Santa Cruz, USA) and MAP-2 (Santa Cruz, USA). Then, the cells were stained with the secondary antibodies Alexa Fluor 488-goat anti-mouse IgG, Alexa Fluor 594-donkey anti-rabbit IgG, Alexa Fluor 488-donkey anti-rabbit IgG and Alexa Fluor 594-donkey anti-mouse IgG (Invitrogen, USA). Then, the cell nuclei were stained with DAPI (4,6-diamidino-2-phenylindole). Then, images were obtained using the Olympus IX71 optical microscope and the MetaMorph software (Molecular Devices, USA).

(35) “Immunohistochemistry” was performed according to the following method.

(36) Tissues were fixed overnight at 4° C. with 4% PFA (Wako, Japan) in PBS. All samples were decalcified with Morse's solution. The samples were dehydrated sequentially with ethanol and xylene, embedded in paraffin (Leica Biosystems, Germany) and then cut to 5 μm thickness. After blocking the cut surface for 15 minutes in 3% hydrogen peroxide, the samples were incubated at 4° C. overnight with primary antibodies. The primary antibodies treated on the cut surface are as follows: mouse monoclonal antibody against HLA class I (Abcam, United Kingdom), goat polyclonal antibody against collagen type II (Santacruz, USA), rabbit polyclonal antibody against osteocalcin (Santacruz, USA), osterix (Abcam, USA), phospho-myosin light chain (pMLC) (Abcam, USA), scleraxis (Antibodies Online, USA), PPARgamma (PPAr) (Santacruz, USA) Runx2 (Novus, USA), DMP1 (Santacruz, USA), vWF (Santacruz, USA) and sclerostin (Santacruz, USA). The used secondary antibodies were Alexa 555 (Invitrogen, USA) and Alexa 488 (Invitrogen, USA) IgG. The immunostained cut surface was counterstained with TO-PRO3 (Invitrogen, USA) to visualize the nuclei. The fluorescence-labeled cut surface was imaged with the Leica DM 5000 microscope (Leica Microsystems, Germany) or a confocal microscope (LSM510; Carl Zeiss, Germany) and analyzed with the Zen software.

Example 6

Flow Cytometry

(37) After separating the hMSSCs of Examples 2.1 and 2.2 into a single cell suspension by treating with trypsin/EDTA and blocking nonspecific binding with 2% BSA in PBS, the cells were reacted with monoclonal antibodies against Sca, CD2, CD3, CD4, CD7, CD8, CD10, CD11b, CD14, CD19, CD20, CD31, CD34, CD44, CD45, CD51, CD56, CD73, CD90, CD105, CD146, CD166, CD235a and CD271 (BD Biosciences, USA) in a buffer solution [1× PBS, 1% BSA and 0.01% sodium azide] and then washed. The cells were reacted with Alexa Fluor 488 secondary mouse-IgGs (Invitrogen, USA), washed and then analyzed using a flow cytometer (FACStar Plus Flowcytometer, BD Biosciences, USA). Normal mouse IgGs (BD Biosciences, USA) were used as negative control group.

Example 7.1

Differentiation of Human Mesenchymal Stem Cell (hMSC) and hMSSC into Osteoblast In Vitro

(38) In order to differentiate the hMSSCs of Examples 2.1 and 2.2 into osteoblasts, the cells were cultured in an osteogenic differentiation medium (StemPro® osteogenic differentiation kit, Life Technologies, USA) under the condition of 37° C. and 5% CO.sub.2 for 14 days. Alkaline phosphatase (Roche, Switzerland) staining and alizarin red S (Sigma, USA) staining were conducted to observe osteogenesis. The differentiation of hMSCs (Lonza, Switzerland) into osteoblasts was also compared in the same manner.

Example 7.2

Differentiation of Human Mesenchymal Stem Cell (hMSC) and hMSSC into Adipocyte In Vitro

(39) In order to differentiate the hMSSCs of Examples 2.1 and 2.2 into adipocytes, the cells were cultured in an adipogenic differentiation medium (StemPro® adipogenic differentiation kit, Life Technologies, USA) under the condition of 37° C. and 5% CO.sub.2 for 14 days. Oil red O (Sigma, USA) staining was conducted to observe adipogenesis. The differentiation of hMSCs (Lonza, Switzerland) into adipocytes was also compared in the same manner.

Example 7.3

Differentiation of Human Mesenchymal Stem Cell (hMSC) and hMSSC into Cartilage Cell In Vitro

(40) In order to differentiate the hMSSCs of Examples 2.1 and 2.2 into cartilage cells, the cells were resuspended in a chondrogenic differentiation medium (StemPro® chondrogenic differentiation kit, Life Technologies, USA) and then centrifuged. For formation of micromass, the formed pellets were resuspended in a differentiation medium to 1×10.sup.5/μL and then 5 μL of the cell solution was dropped at the center of a 96-well plate. After incubating the micromass for 2 hours under a high-humidity condition and adding a heated chondrogenic differentiation medium, incubation was performed in an incubator under the condition of 5% CO.sub.2 and 37° C. The culture medium was re-feeded with 3- to 4-day intervals. 14 days later, the chondrogenic pellets were stained with Alcian blue. The differentiation of hMSCs (Lonza, Switzerland) into cartilage cells was also compared in the same manner.

Example 8.1

Differentiation Capacity of hMSSC into Endothelial Cell In Vitro

(41) It was investigated whether the hMSSCs of Example 2.1 are differentiated into endothelial cells (ECs). The hMSSCs were differentiated by culturing with a medium for inducing differentiation into an EC (endothelial growth medium (EGM)-2 (Lonza, Walkersville, Md., USA) supplemented with 50 ng/mL VEGF (vascular endothelial growth factor: ProSpec, Rehovot, Israel) and 10 ng/mL bFGF (basic fibroblast growth factor; ProSpec, Rehovot, Israel) for 6 days. The differentiation was confirmed by immunocytochemistry.

Example 8.2

Differentiation Capacity of hMSSC into Skeletal Muscle Cell In Vitro

(42) It was investigated whether the hMSSCs of Examples 2.1 and 2.2 are differentiated into skeletal muscle cells. The hMSSCs were differentiated by culturing with a skeletal muscle differentiation medium (DMEM supplemented with 2% B27) for 2 weeks on a Matrigel-coated cover slip. The differentiation was confirmed by immunocytochemistry.

Example 9

Induction of Differentiation from hMSSC to Nerve Cell In Vitro

(43) For differentiation into nerve cells, the hMSSCs of Example 2.1 were plated on a polyornithine- and laminin-coated culture dish. 2 days later, the culture medium was exchanged with a medium for inducing differentiation into a nerve (Neurobasal medium containing 2% B27, 2 mM GlutaMAX and antibiotics). From day 7, 0.5 mM dibutyl cAMP (Sigma, USA) was added every day for 3 days. As a control group, human neural stem cells differentiated from H9 hESCs (Gibco, USA) were differentiated into nerve cells in the same manner. The differentiation was confirmed by immunocytochemistry.

Example 10.1

Differentiation Capacity of hMSSC in Mouse Kidney

(44) In order to measure the differentiation capacity of the hMSSCs of Example 2.1 in mouse kidney, the hMSSCs were cultured with a MSCGM-CD (Lonza, Switzerland) medium for 2-5 passages and the hMSSCs (2×10.sup.5 cells) were cultured in an agarose gel well with DMEM+20% FBS for 2 days to form cell aggregates, which were transplanted into the kidney capsule of Balb/c nude mouse. Immunohistochemistry and immunohistochemical staining were performed 4 weeks after the transplantation.

Example 10.2

Differentiation Capacity of hMSSC in Mouse Hypoderm

(45) In order to measure the differentiation capacity of the hMSSCs of Example 2.1 in mouse hypoderm, the hMSSCs were cultured with a MSCGM-CD (Lonza, Switzerland) medium for 2-5 passages and the hMSSCs (2×10.sup.5 cells) were loaded in fibrin glue (Greenplast®, Green Cross, Korea) to which 1 μg/mL hyaluronic acid (Sigma, USA) was added and then transplanted into the hypoderm of Balb/c nude mouse. Immunohistochemistry and immunohistochemical staining were performed 4 weeks after the transplantation.

Example 11.1

Osteogenesis Test Using hMSC

(46) For analysis of osteogenesis of hMSCs in a thighbone fracture model, hMSCs (Lonza, Switzerland) were cultured with a MSCGM-CD (Lonza, Switzerland) medium for 7 passages and then absorbed into a collagen membrane (SK Bioland, Korea) cut to a size of 1 mm×1 mm. After perforating one tibia of a 6-week-old Balb/c nude mouse about 1 mm using a drill (Bosch Professional, Germany), the hMSCs absorbed in the collagen membrane were inserted into the fracture site of the mouse. Every two weeks, the mouse was anesthetized and micro-CT (Skyscan 1076, Antwerp, Belgium) images were obtained for the fracture site. Immunohistochemistry and immunohistochemical staining were performed 6 weeks later.

Example 11.2

Osteogenesis Test Using hMSSC

(47) For analysis of osteogenesis of hMSSC in a thighbone fracture model, the hMSSCs of Example 2.1 were cultured with a MSCGM-CD (Lonza, Switzerland) medium for 2-5 passages and then absorbed into a collagen membrane (SK Bioland, Korea) cut to a size of 1 mm×1 mm. After perforating one tibia of a 6-week-old Balb/c nude mouse about 1 mm using a drill (Bosch Professional, Germany), the hMSSCs absorbed in the collagen membrane were inserted into the fracture site of the mouse. Every two weeks, the mouse was anesthetized and micro-CT (Skyscan 1076, Antwerp, Belgium) images were obtained for the fracture site. Immunohistochemistry and immunohistochemical staining were performed 6 weeks later.

Example 12

Micro-CT

(48) The bone formed in the kidney into which the hMSSCs were transplanted in Example 10.1 was scanned by micro-CT (Skyscan 1076, Antwerp, Belgium) to obtain 3D CT (computed tomography) images. Then, the data were digitalized with a frame grabber and the resulting images were transmitted to a computer using the Comprehensive TeX Archive Network (CTAN) topographic reconstruction software.

Example 13

Measurement of scx, Runx2 and MYH9 mRNA Expression Levels

(49) RNAs were extracted from the transplant of the hMSSCs of Example 2.1 in the kidney using 500 μL of Trizol (Life Technologies, USA) according to the manufacturer's protocol. After treating the transplant of the hMSSCs in the kidney with DNAse (RQ1 DNase, Promega, USA), 500 ng of RNAs were reversely transcribed to cDNAs using oligo-d(T) and random hexamers according to the Superscript III RT (Life Technologies, USA) first-strand cDNA synthesis protocol. qRT-PCR was conducted on the StepOne Plus PCR cycler (Applied Biosystems) using SYBR green (Applied Biosystems, Foster City, Calif.). mRNA expression data were analyzed using the ΔΔCT method and normalized with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) for gene detection. The primers necessary for the qRT-PCR were purchased from Qiagen (USA). As a control group, RNAs were extracted from hMSSCs and qRT-PCR was conducted in the same manner.

(50) Experimental Results

Test Example 1

Confirmation of Induction of Differentiation of hMSSC Derived from hESC

(51) Aging Marker

(52) The differentiation from hESCs to hMSSCs was induced as described in Example 2 and the morphological change of the induced hMSSCs was observed. The result is shown in FIG. 1A. As seen from FIG. 1A, it was confirmed that the undifferentiated single H9 hESCs were differentiated into cells with fibroblast morphology within 7 passages. They grew with similar morphologies for 10 passages or longer, from passage 7 to passage 17, and showed a positive response to staining with the aging marker β-galactosidase since passage 19, suggesting that aging was progressed.

(53) Confirmation of Pluripotency Marker by Immunofluorescence Method

(54) The expression of pluripotency markers in the hMSSCs after 7 passages or longer since the induction from the hESCs was observed by the immunofluorescence method. The result is shown in FIG. 1B. For comparison, the expression of pluripotency markers in H9 hESCs was investigated by the immunofluorescence method.

(55) As seen from FIG. 1B, the H9 hESCs were positive for all of OCT4, NANOG, SOX2 and LIN28, suggesting that they have pluripotency. In contrast, the hMSSCs induced from the H9 hESCs were negative for OCT4, NANOG, SOX2 and LIN28.

(56) Confirmation of Pluripotency, Ectodermal, Mesodermal and Endodermal Markers Through RNA Sequencing

(57) The expression of pluripotency, ectodermal, mesodermal and endodermal markers in hESCs, hMSCs and hMSSCs at passages 7 and 17 was investigated through RNA sequencing. The result is shown in FIG. 1C. The expression of the mRNAs of the pluripotency markers TDGF, NANOG, POU5F1, SOX2, DPPA4, LEFTY1, GDF3, etc. was confirmed in the H9 hESCs (hESC-1, hESC-2). In contrast, for the hMSSCs induced from the H9 hESCs, the expression of the pluripotency marker DPPA4 was observed but the expression of the pluripotency markers TDGF, NANOG, POU5F1, LEFTY1 and GDF3 was not observed. The expression level of DPPA4 was comparable to that in the H9 hESCs.

(58) The expression of DPPA4 was not observed in the human mesenchymal stem cells. In addition, the hMSSCs were positive for the ectodermal marker NES, were positive for most mesodermal markers except for DES and the early mesodermal markers T and nodal and were negative for most endodermal markers. In particular, NES was not expressed in the mesenchymal stem cells.

(59) Confirmation of Mesenchymal Stem Cell Markers Through Expression of Cell Surface Antigens

(60) The expression of antigens on the surface of hMSSCs was measured as seen from FIG. 1D. When the expression of mesenchymal stem cell-specific cell surface antigens was investigated, the expression of the mesenchymal stem cell markers CD44, CD51, CD73, CD105, CD146 and CD166 was observed in the hMSSCs but the expression of the mesenchymal stem cell markers CD90 and CD271 was not observed. In addition, the expression of the vascular cell surface markers CD2, CD3, CD7, CD8, CD11 b, CD14, CD19, CD20, CD31, CD34 and CD56 was not observed but the expression of the pre-B cell marker CD10 was observed.

(61) Confirmation of Other Cell-Specific Markers

(62) The expression of various tissue-specific markers was analyzed to investigate the characteristics of hMSSCs as shown in FIG. 1F. The mesodermal marker alpha smooth muscle actin (a-SMA), the neuroectodermal marker Pax6, the myogenic satellite marker Pax7, the intestinal stem cell marker LGR5, etc. were expressed, whereas the chondrocyte marker SOX9, the myoblast marker MyoD, etc. were not expressed. This suggests that the hMSSCs are progenitor cells prior to differentiation into cartilage cells and muscle cells.

Test Example 2

Differentiation Capacity of hMSSC In Vitro

(63) In-vitro osteogenesis, chondrogenesis and adipogenesis were tested for hMSCs and the hMSSCs of Example 2.1 (Example 7) and the result is shown in FIG. 2A. From FIG. 2A, it was confirmed that the hMSCs could be differentiated into bone, cartilage and fat in vitro. Meanwhile, the hMSSCs were differentiated into bone and cartilage but were hardly differentiated into fat under the same conditions in vitro. That is to say, the cells were found to be functionally different from the mesenchymal stem cells.

(64) Differentiability into Skeletal Muscle

(65) It was investigated whether the hMSSCs of Test Example 1 has the potential to be differentiate into skeletal muscle.

(66) The hMSSCs were cultured for 2 weeks in a medium for inducing differentiation into skeletal muscle (DMEM containing 2% B27) on a Matrigel-coated cover slip and then immunofluorescence assay was performed for the skeletal muscle marker MYH9. The result is shown in FIG. 2B. C2C12 cells were used as a control group. As seen from FIG. 2B, it was confirmed that the skeletal muscle-specific marker MYH9 was expressed when the hMSSCs were cultured in the skeletal muscle differentiation medium, suggesting that the hMSSCs have the potential to be differentiate into skeletal muscle.

(67) Differentiability into Endothelial Cell

(68) It was investigated whether the hMSSCs of Test Example 1 has the potential to be differentiate into endothelial cells.

(69) The hMSSCs were cultured for 6 days in a medium for inducing differentiation into an EC (endothelial growth medium (EGM)-2 (Lonza, Walkersville, Md.)) supplemented with 50 ng/mL VEGF (vascular endothelial growth factor: ProSpec, Rehovot, Israel) and 10 ng/mL bFGF (basic fibroblast growth factor; ProSpec) and then immunofluorescence assay was performed for the endothelial cell markers CD31 and VE-cadherin. The result is shown in FIGS. 2C and 2D. HUVECs were used as a positive control group for endothelial cell differentiation. As seen from FIG. 2C and FIG. 2D, the expression of CD31 and VE-cadherin was not observed in the hMSSCs, suggesting that the hMSSCs lack the potential to be differentiate into endothelial cells. In contrast, expression of the markers were observed in the control group HUVECs.

(70) Differentiability into Nerve Cell

(71) The hMSSCs were incubated for 7 days in a medium for inducing differentiation into a nerve (Neurobasal medium containing 2% B27, 2 mM GlutaMAX and antibiotics) and then cultured for 3 days while adding 0.5 mM dibutyl cAMP (Sigma) every day. Then, immunofluorescence assay was performed for the nerve cell differentiation marker MAP2. The result is shown in FIG. 2E. NSCs (neuronal stem cells) were used as a positive control group for nerve cell differentiation. As seen from FIG. 2E, the cell morphology of the NSCs was changed to that of nerve cells and the expression of the nerve cell-specific marker MAP2 was observed, suggesting that the cells were differentiated into nerve cells. In contrast, the hMSSCs showed no change in cell morphology and the expression of MAP2 was not observed, suggesting that they lack the potential to be differentiate into nerve cells.

(72) Although the hMSSCs were positive for the ectodermal marker NES as confirmed in Test Example 1, they were not differentiated into nerve cells. It was confirmed that the hMSSC can be differentiated into the mesoderm, more particularly to bone, cartilage and muscle.

Test Example 3

Confirmation of Differentiation of hMSSC into Bone, Cartilage, Muscle, Fat and Tendon In Vivo

(73) In order to measure the differentiability of the hMSSCs induced in the same manner as in Example 2 in vivo, the hMSSCs were transplanted into the kidney (Example 10.1) and hypoderm (Example 10.2) of an immune-deficient mouse. After transplanting the hMSSCs into mouse kidney and staining tissues with H&E 3-4 weeks later, immunofluorescence staining was performed for bone-, muscle-, fat- and tendon-specific markers and the cell nuclei were counterstained with TO-PRO3. The result is shown in FIG. 3A and FIG. 3B.

(74) FIG. 3A shows images obtained 4 weeks after culturing the hMSSCs in a MSCGM-CD (Lonza, Switzerland) medium for 2-5 passages and transplanting them into the kidney. The H&E staining result shows that muscle, fat and tendon were formed well in the kidney (FIG. 3A-a). When the differentiated muscle tissues were analyzed, the differentiation into skeletal muscle was observed but the differentiation into smooth muscle was not observed. In contrast, when human MSCs were transplanted under the same condition, muscle, fat, tendon, etc. were not formed at all (data not shown). When immunohistochemical assay was performed, it was confirmed that each differentiated tissue was positive for the muscle marker phospho-myosin light chain (pMLC), the adipose marker PPARgamma (PPAr), the tendon marker sleraxis (Scx), etc. and was also positive for the human cell marker hLA (human leukocyte antigen). From this, it can be seen that transplanted hMSSCs were differentiated into muscle, fat and tendon cells (This is contrary to the in-vitro test result showing no differentiation into fat) (FIG. 3A-b).

(75) FIG. 3B-a shows a result of micro-CT scanning showing that hard tissue, or bone, was formed at the site where the hMSSC was transplanted into the kidney.

(76) FIGS. 3B-b and 3B-c show a result of confirming bone formation by H&E and pentachrome staining. It can be seen that the transplanted hMSSCs were differentiated into bone in the kidney capsule.

(77) FIG. 3B-d shows the immunohistochemical assay at the transplanted site. It was confirmed that the cells in the tissue were positive for the human cell marker hLA (human leukocyte antigen), the bone markers Osx (osterix), Runx2, DMP1, OCN (osteocalin), etc. and the vascular marker vWF, suggesting that bone was confirmed. Therefore, it can be seen that the transplanted hMSSCs were differentiated into bone.

(78) FIG. 3C shows that the hMSSCs transplanted into mouse hypoderm by loading in fibrin glue to which hyaluronic acid was added were differentiated into cartilage cells. The cartilage formation was confirmed by H&E and toluidine blue staining.

(79) Taken together, it was confirmed that the hMSSCs of the present disclosure can be differentiated into cartilage, muscle, tendon and bone at the transplanted site and have superior differentiation capacity.

Test Example 5

Confirmation of Fracture Recovery Effect of hMSSC

(80) In order to confirm the fracture recovery of hMSSCs induced in the same manner as in Example 2, osteogenesis test was performed as in Example 11. The result is shown in FIGS. 4A-4B.

(81) It was confirmed that, when the hMSCs were transplanted into a fracture site in a thighbone fracture model, bone was formed about 6 weeks later at the fracture site. However, because the osteogenic site was positive for the bone marker Runx2 but negative for the human cell marker hLA, it was estimated that the osteogenesis was not by the transplanted hMSCs but by the mouse cells (FIG. 4A). In contrast, when hMSSCs were transplanted under the same condition, bone was formed about 6 weeks later at the fracture site, with the osteogenic site being positive for Runx2 and positive for the human cell marker hLA. This suggests that the bone formation was owing to the differentiation of the hMSSCs (FIG. 4B).

Test Example 6

Induction of Differentiation from hiPSC into hMSSC and Characterization of Induced hiPSC

(82) hiPSCs (human induced pluripotent stem cells) were prepared by reprogramming embryonic IMR90 fibroblasts by overexpressing OCT4, KLF4, SOX2 and MYC using Sendai virus according to the method developed by Hasegawa et al. (Fusaki et al., 2009).

(83) iPS-hMSSCs were obtained by inducing hMSSCs from hiPSCs in the same manner as in Example 2. The expression level of the pluripotency markers Oct4, Nanog, Sox2 and Lin28 in the iPS-hMSSCs was investigated by immunofluorescence assay and RT-PCR. The result is shown in FIG. 5A.

(84) As seen from FIG. 5A, iPS cells were positive for OCT4, NANOG, SOX2 and LIN28, suggesting that the cells have pluripotency. In contrast, the iPS-hMSSCs were negative for the pluripotency markers OCT4, NANOG, SOX2 and LIN28.

(85) FIG. 5B shows a result of measuring the expression of surface antigens for the iPS-hMSSCs. When the expression of mesenchymal stem cell-specific cell surface antigens was investigated, it was confirmed that, among the mesenchymal stem cell markers, CD44, CD51, CD73, CD105, CD146 and CD166 were expressed in the iPS-hMSSCs, but CD90 and CD271 were not expressed in the iPS-hMSSCs. In addition, the pre-B cell marker CD10 was expressed whereas the vascular cell surface markers CD2, CD3, CD7, CD8, CD14, CD20 and CD56 were not expressed.

(86) Also, the osteogenesis, chondrogenesis and adipogenesis of the iPS-hMSSCs were evaluated in the same manner as in Test Example 2. The result is shown in FIG. 5C. As seen from FIG. 5C, it was confirmed that the hMSSCs induced from the hiPSCs were differentiated into bone and cartilage in vitro but were hardly differentiated into fat.

(87) In addition, the iPS-hMSSCs were cultured for 2 weeks in a medium for inducing differentiation into skeletal muscle (DMEM containing 2% B27) on a Matrigel-coated cover slip and then immunofluorescence assay was performed for the skeletal muscle marker MYH9. The result is shown in FIG. 5D. C2C12 cells were used as a control group. As seen from FIG. 5D, it was confirmed that the hMSSCs induced from the hiPSCs have the potential to be differentiate into skeletal muscle.

(88) Taken together, it was confirmed that the hMSSCs induced from the hiPSCs have the same characteristics as the hMSSCs induced from the hECSs, suggesting that hMSSCs can be obtained using hiPSCs instead of hECSs.

Test Example 7

Differentiability of hMSSC Induced from hiPSC In Vivo

(89) Transplantation into Kidney

(90) After transplanting the hMSSCs of Test Example 6 into mouse kidney, the tissue was stained with H&E 3-4 weeks later. It was confirmed that muscle, fat and tendon were formed in the kidney. The immunohistochemical assay result for the transplanted site was positive for the muscle marker phospho-myosin light chain (pMLC), the adipose marker PPARgamma (PPAr), the tendon marker sleraxis (Scx), etc. and also positive for the human cell marker hLA (human leukocyte antigen). Also, the result was positive for the bone markers Osx (osterix), Runx2, DMP1, OCN (osteocalin), etc. Through this, it was confirmed that the hMSSCs induced from the iPSCs can be differentiated into muscle, fat, tendon and bone.

(91) Transplantation into Hypoderm

(92) When the hMSSCs of Test Example 6 were transplanted into mouse hypoderm by loading in fibrin glue to which hyaluronic acid was added, it was confirmed through H&E and toluidine blue staining that the hMSSCs can be differentiated into cartilage.

Comparative Example 1

Comparison of Differentiation Capacity of Noggin-Containing MSSC Medium and Conditioned Medium-Containing CM Medium

(93) The differentiation capacity of a medium (hereinafter, “CM medium”) obtained by replacing the human noggin (Life Technologies), i.e., the constitutional ingredient 1) of the seven constitutional ingredients of the MSSC medium of Example 2, with a conditioned medium (a culture supernatant obtained after culturing CF1 cells with a medium obtained by replacing DMEM/F12 in a complete medium with knockout DMEM (supplemented with 20% knockout serum replacement (Invitrogen, USA), 1 mM glutamine, 1% nonessential amino acids (Invitrogen, USA), 0.1 mM β-mercaptoethanol, 0.1% penicillin-streptomycin and 5 mg/mL bovine serum albumin)) (the remaining constitutional ingredients 2)-7) are identical) was compared with that of the MSSC medium.

(94) Noggin is generally used to maintain the characteristics of hESCs during culturing (Chaturvedi G, Simone P D, Ain R, Soares M J, Wolfe M W. Noggin maintains pluripotency of human embryonic stem cells grown on Matrigel. Cell Prolif. 2009 August; 42(4): 425-33). Contrarily to the previously known mechanism, it significantly increased the tendency toward the mesoderm. As can be seen from Table 1, the tendency for osteogenic differentiation was increased 10 times or greater when noggin was contained, as compared when the CM medium was used.

(95) TABLE-US-00001 TABLE 1 Differentiation tendency of MSSC medium vs. CM medium (number of observations out of 20 differentiations) Medium Bone Muscle Tendon Fat CM medium  1/20 20/20  2/20  2/20 Noggin-containing 15/20 20/20 10/20 12/20 medium

(96) Also, the expression level of CD44 was compared for the two media. After inducing differentiation using the CM-containing medium (CM medium) and the noggin-containing medium (MSSC medium), the expression level of CD44 was measured in the same manner as in Example 6. As a result, it was confirmed that the expression level of CD44 was increased remarkably when the noggin-containing MSSC medium was used as compared to when the CM medium was used (FIG. 6).

(97) During osteogenic differentiation, the formation of endochondral bone occurs only after chondrogenesis. CD44 is known to play an essential role in chondrogenesis (Wu S C, Chen C H, Chang J K, Fu Y C, Wang C K, Eswaramoorthy R, Lin Y S, Wang Y H, Lin S Y, Wang G J, Ho M L: Hyaluronan initiates chondrogenesis mainly via cd44 in human adipose-derived stem cells. J Appl Physiol (1985) 2013; 114: 1610-1618). From the above results, it can be seen that use of the MSSC medium rather than the CM medium is suitable for osteogenic differentiation.

(98) When hMSSCs were transplanted into the kidney, the cells differentiated by the hMMSC medium showed 1-2 weeks faster differentiation as compared to the cells differentiated by the CM medium. The difference in differentiation speed when the CM medium was used and when the hMMSC medium was used is shown in Table 2.

(99) TABLE-US-00002 TABLE 2 Differentiation speed of MSSC medium vs. CM medium (increased mRNA level as compared to before transplantation of hMSSC) mRNA level Week 1 Week 2 Week 3 Week 4 MYH9 CM 1.3 ± 0.1 2.1 ± 0.1 5.1 ± 0.3 12.5 ± 3.1 MSSC 2.2 ± 0.3 4.4 ± 0.4 20.1 ± 3.1  23.1 ± 3.4 Runx2 CM 1.2 ± 0.3 1.8 ± 0.3 3.6 ± 0.3  6.5 ± 3.1 MSSC 2.1 ± 0.2 4.3 ± 0.3 7.1 ± 0.3 13.3 ± 3.1 Scx CM 1.3 ± 0.2 2.3 ± 1.2 5.2 ± 1.3 10.7 ± 2.2 MSSC 2.1 ± 0.2 4.7 ± 1.5 12.1 ± 0.3  16.5 ± 2.9

Comparative Example 2

Comparison of Synergistic Effect for Combinations of Constitutional Ingredients of MSSC Medium

(100) The differentiation capacity of the MSSC medium of Example 2 not containing one of the constitutional ingredients 1)-6) was compared with that of the MSSC medium. As a result, it was confirmed that differentiation into cartilage (Alcian blue) or bone (ALP and Alizarin red S) was not achieved well when one of the constitutional ingredients 1)-6) was absent (FIG. 7, Table 3).

(101) TABLE-US-00003 TABLE 3 Comparison of differentiation capacity of MSSC medium vs. medium deficient in one constitutional ingredient TGF-β/ activin/ nodal ERK Wnt FGF-2 Constitutional signaling signaling signaling signaling ingredient of inhibitor hLIF inhibitor activator Noggin activator MSSC medium 7 ingredients (−) (−) (−) (−) (−) (−) 1) Noggin ◯ ◯ ◯ ◯ ◯ X ◯ 2) LIF ◯ ◯ X ◯ ◯ ◯ ◯ 3) FGF-2 ◯ ◯ ◯ ◯ ◯ ◯ X signaling activator 4) Wnt ◯ ◯ ◯ ◯ X ◯ ◯ signaling activator 5) ERK ◯ ◯ ◯ X ◯ ◯ ◯ signaling inhibitor 6) TGF-β/ ◯ X ◯ ◯ ◯ ◯ ◯ activin/ nodal signaling inhibitor 7) Others ◯ ◯ ◯ ◯ ◯ ◯ ◯ Remarks Differentiated Not Not Differentiated Differentiated MSSCs MSSCs into muscle differentiated differentiated into cartilage into bone was were not were not (including into muscle into cartilage and bone was inhibited induced induced adipose), or cartilage inhibited cartilage and bone

(102) Those skilled in the art will appreciate that the conceptions and specific embodiments disclosed in the foregoing description may be readily utilized as a basis for modifying or designing other embodiments for carrying out the same purposes of the present disclosure. Those skilled in the art will also appreciate that such equivalent embodiments do not depart from the spirit and scope of the disclosure as set forth in the appended claims.