COMPOSITION AND METHOD FOR INDUCING DIFFERENTIATION INTO MYELOID CELLS, AND USE THEREOF

Abstract

Provided are a composition and method for inducing direct conversion from a somatic cell into a myeloid cell and use thereof, in which differentiation from a somatic cell into a myeloid cell can be efficiently induced through the expression of a single direct conversion inducer without undergoing the pluripotency stage of induced pluripotent stem cells, and thus, the composition can be widely used as an effective preventive and therapeutic agent for immune diseases.

Claims

1. A composition for inducing direct conversion from a somatic cell into a myeloid cell, the composition comprising at least one selected from the group consisting of: (1) a friend leukemia (virus) integration 1 (FLI1, ETS transcription factor) protein; (2) a nucleic acid molecule encoding the protein; and (3) a vector into which the nucleic acid molecule is introduced.

2. The composition of claim 1, wherein the myeloid cell comprises at least one selected from the group consisting of a monocyte progenitor cell, a monocyte, a macrophage, a dendritic cell, a megakaryocyte, and a platelet.

3. The composition of claim 1, wherein the somatic cell comprises at least one selected from the group consisting of a fibroblast, an epithelial cell, a muscle cell, a nerve cell, a hair cell, a hair root cell, a hair follicle cell, an oral epithelial cell, a somatic cell extracted from urine, a gastric mucosal cell, a goblet cell, a G cell, a B cell, a pericyte, an astrocyte, a blood cell, a neural stem cell, an oligodendrocyte progenitor cell, a hematopoietic stem cell, an umbilical cord blood stem cell, and a mesenchymal stem cell.

4. The composition of claim 1, wherein the vector comprises at least one selected from the group consisting of a plasmid vector, a cosmid vector, a viral vector, a lentiviral vector, a retrovirus vector, a human immunodeficiency virus (HIV) vector, a murineleukemia virus (MLV) vector, an avian sarcoma/leukosis (ASLV) vector, a spleen necrosis virus (SNV) vector, a rous sarcoma virus (RSV) vector, a mouse mammary tumor virus (MMTV) vector, an adenovirus vector, an adeno-associated virus vector, a Herpes simplex virus vector, and an episomal vector.

5. A method for direct conversion of a somatic cell into a myeloid cell in vitro, the method comprising bringing or inserting, into contact with or into a somatic cell, a composition comprising at least one selected from the group consisting of: (1) a friend leukemia virus integration 1 (FLI1) protein; (2) a nucleic acid molecule encoding the protein; and (3) a vector into which the nucleic acid molecule is introduced.

6. A myeloid cell induced through direct conversion by bringing or inserting, into contact with or into a somatic cell, a composition comprising at least one selected from the group consisting of: (1) a friend leukemia virus integration 1 (FLI1) protein; (2) a nucleic acid molecule encoding the protein; and (3) a vector into which the nucleic acid molecule is introduced.

7. A pharmaceutical composition for the prevention or treatment of an immune disease, the pharmaceutical composition comprising the composition of claim 1 as an active ingredient.

8. The pharmaceutical composition of claim 7, wherein the immune disease comprises at least one selected from the group consisting of Crohn's disease, erythema, atopy, rheumatoid arthritis, Hashimoto's thyroiditis, malignant anemia, Edison's disease, type 1 diabetes, lupus, chronic fatigue syndrome, fibromyalgia, hypothyroidism, hyperthyroidism, scleroderma, Bahcet's disease, inflammatory bowel disease, multiple sclerosis, Alzheimer's disease, Parkinson's disease, myasthenia gravis, Meniere's syndrome, Guilian-Barre syndrome, Sjogren's syndrome, vitiligo, endometriosis, psoriasis, vitiligo, systemic scleroderma, asthma, and ulcerative colitis.

9. A cell therapeutic agent for the prevention or treatment of an immune disease, the cell therapeutic agent comprising the myeloid cell induced through direct conversion of claim 6 as an active ingredient.

10. A composition for screening for a drug for the prevention or treatment of an immune disease, the composition comprising the myeloid cell induced through direct conversion of claim 6 as an active ingredient.

11. Use of the composition according to any one of claims 1 to 4 for the preparation of a drug for the prevention or treatment of an immune disease.

12. A method for the prevention or treatment of an immune disease, the method comprising administering a therapeutically effective amount of the pharmaceutical composition of claim 7 to a subject.

Description

BRIEF DESCRIPTION OF DRAWINGS

[0064] FIG. 1 illustrates a view showing a process of inducing monocyte progenitor cells, according to an embodiment (FIG. 1A) and the morphology of cells obtained in the process of inducing monocyte progenitor cells (FIG. 1B).

[0065] FIG. 2 illustrates the results of flow cytometric analysis of induced monocyte progenitor cells (iMac) according to an embodiment, mouse fibroblasts, and bone marrow-derived monocyte progenitor cells (BMDM), against CD11b and F4/80 antibodies.

[0066] FIG. 3 illustrates the results of confirming the expression of CD45, CX3CR1, CD11b, and IBA-1 after immunocytochemical staining was performed on induced monocyte progenitor cells according to an embodiment.

[0067] FIG. 4 illustrates the results of confirming the phagocytosis ability of induced monocyte progenitor cells according to an embodiment.

[0068] FIG. 5 illustrates changes in cell shape after activation of induced monocyte progenitor cells according to an embodiment (FIG. 5A) and the results of confirming changes in expression levels of immune response-related genes (FIG. 5B).

[0069] FIG. 6 illustrates changes in survival rate and body weight after transplantation of induced monocyte progenitor cells according to an embodiment or activated macrophages to an inflammatory bowel disease animal model (FIG. 6A), disease activity index (FIG. 6B), colon length (FIG. 6C), histological score (FIG. 6D), and H&E staining of intestinal tissue (FIG. 6E).

BEST MODE

[0070] Hereinafter, the present disclosure will be described in detail with reference to experimental examples and examples to aid the understanding of the present disclosure. However, the following experimental examples and examples are illustrative purposes only and are not intended to limit the scope of the present disclosure. The examples and experimental examples of the present disclosure are provided to completely explain the present disclosure to those of ordinary skill in the art.

Example 1. Cloning and Packaging of Lentiviral Plasmid

[0071] A transcription factor (FLI1) was amplified by a polymerase chain reaction (PCR) using cDNA of HepG2. Subsequently, the amplified FLI1 was introduced into a lentiviral vector and confirmed by sequencing. The packaging of lentivirus was performed by adding a lentiviral transfer plasmid, a packaging plasmid (psPAX2), and an envelope plasmid (VSV-G) in a ratio of 3:2:1 by using an X-tremeGENE 9 DNA transfection reagent (Roche, 06365787001). 293T cells (Thermo Fisher Scientific) at a confluency of 50% were treated with a mixture of the total volume was made to 200 μl by adding DMEM (Gibco, 10313-021), and transformed for 48 hours. Thereafter, the cell culture medium was centrifuged at 80,000 g in an ultracentrifuge for 1.5 hours, and then the lentivirus pellet obtained from the centrifuged supernatant was diluted in 1 ml of DMEM.

Example 2. RNA Extraction and cDNA Synthesis

[0072] Total RNA of the cell lysate was extracted with RiboEX (Geneall, 301-001). RNA extraction was performed according to the manufacturer's protocol. cDNA was synthesized with M-MuLV-reverse transcriptase (NEB, M0253L) by adding 500 ng of RNA and oligo-dT primer to a total of 20 μl of the reaction mixture.

Example 3. Immunocytochemical Staining

[0073] Cells were fixed with 4% paraformaldehyde (Tech & Innovation, BPP-9004) at room temperature for 10 minutes. The fixed cells were washed three times with Dulbecco's phosphate-buffered saline (DPBS, Corning, 21-031-CV). Subsequently, the cells were permeabilized by treatment with DBPS containing 0.1% Triton X-100 (Sigma, T9284) at room temperature for 10 minutes. The cells were washed three times with DPBS, followed by reacting with DPBS containing 4% FBS at room temperature for 1 hour, to block non-specific binding. The cells were cultured with primary antibodies for 1 hour at room temperature, and then washed three times with DPBS containing 0.05% Tween-20 (Sigma, P7949). Thereafter, the cells were treated with secondary fluorescent antibodies and incubated in the dark for 1 hour. If double staining is required, an additional blocking step was performed for 30 minutes before treatment with primary antibodies according to the above process.

Example 4. Confirmation of Immune Response and Inflammatory Response Through Quantitative Real-Time PCR

[0074] DNA-free total RNA was extracted using an RNeasy mini kit (Qiagen). A total of 500 ng of RNA per reaction was used to synthesize cDNA with SuperScript® III reverse transcriptase (Invitrogen). The synthesized cDNA had a total volume of 20 μl and was used as a template by LightCycler 480 SYBR Green I Mastermix (Roche). For immune response-related genes, the experiment was repeated three times, and the genes were normalized to the housekeeping gene GAPDH. Gene expression was measured by the Ct value calculation method, and all experiments were performed according to the manufacturer's protocol.

Example 5. Production of Inflammatory Bowel Disease (IBD) Animal Model and Cell Transplantation

[0075] A colitis animal model was produced by oral administration of dextran sulfate sodium (DSS) to 4-week-old mice. After ingestion of DSS for 5 days to induce acute colitis, induced monocyte progenitor cells or activated M1/M2 macrophages were transplanted into the colon of mice.

Experimental Example 1. Production of Induced Monocyte Progenitor Cells by Direct Conversion Factor

[0076] For direction conversion from somatic cells into monocyte progenitor cells, mouse fibroblasts were dispensed into a culture medium at a density of 1.5×10.sup.4 cells, and after 24 hours, the fibroblasts were transformed with the transcription factor FLI1 through the lentivirus expression system produced according to Example 1. After 24 hours, the medium composition was replaced with a monocyte progenitor cell induction medium to induce differentiation into monocyte progenitor cells. The monocyte progenitor cells induced by mechanical separation of the monocyte progenitor cell population were picked and subcultured. A schematic view showing the processes of the experiment is illustrated in FIG. 1A, and the histological morphology of cells obtained by culturing the induced monocyte progenitor cells is illustrated in FIG. 1B.

[0077] As illustrated in FIG. 1B, it was confirmed that cell aggregates were formed about 36 days after the transformation with lentivirus, and the typical morphology of monocyte progenitor cells appeared after separation and culturing. Through these results, it was confirmed that, when fibroblasts as somatic cells were treated with FLI1 to induce ectopic expression of the factors, monocyte progenitor cells could be effectively produced. In particular, it was confirmed that the monocyte progenitor cells induced by the above process showed the histological morphology shown in the actual monocyte progenitor cells.

Experimental Example 2. Characterization of Induced Monocyte Progenitor Cells

Experimental Example 2.1. Flow Cytometry

[0078] To confirm whether the monocyte progenitor cells (iMac) induced according to Experimental Example 1 show not only the histological morphology but also the characteristics of actual monocyte progenitor cells, fluorescence activated cell sorter (FACS) analysis was performed. Specifically, to confirm whether the induced monocyte progenitor cells obtained according to Experimental Example 1 express CD11b and F4/80, which are monocyte progenitor cell-specific markers, FACS analysis using CD11b and F4/80 antibodies was performed on each of fibroblasts and bone marrow-derived monocyte progenitor cells, and the results thereof are illustrated in FIG. 2.

[0079] As illustrated in FIG. 2, it was confirmed that the induced monocyte progenitor cells had a markedly different expression pattern from fibroblasts, and the monocyte progenitor cell markers CD11b and F4/80, which were not expressed in the fibroblasts, were strongly expressed in the induced monocyte progenitor cells, as in the bone marrow-derived monocyte progenitor cells. The expression levels of the CD11b and F4/80 markers in the induced monocyte progenitor cells were confirmed to be 85.4, which is similar to the expression levels thereof, i.e., 97.2, in the bone marrow-derived monocyte progenitor cells.

[0080] Through this, it was confirmed that the method described in Experimental Example 1 could efficiently induce monocyte progenitor cells.

Experimental Example 2.2. Immunocytochemical Staining

[0081] To confirm whether the monocyte progenitor cells induced according to Experimental Example 1 express CD45, CX3CR1, CD11b, and IBA-1, which are monocyte progenitor cell-specific markers, immunocytochemical staining was performed according to the method described in Example 3 and the results thereof are illustrated in FIG. 3.

[0082] As illustrated in FIG. 3, it was confirmed that the induced monocyte progenitor cells expressed all of CD45, CX3CR1, CD11b, and IBA-1.

[0083] Through this, it was confirmed that the monocyte progenitor cells induced through direct conversion from somatic cells by using the method described in Experimental Example 1 had not only the morphology but also the actual characteristics of monocyte progenitor cells.

Experimental Example 3. Confirmation of Phagocytosis Ability of Induced Monocyte Progenitor Cells

[0084] To confirm whether the monocyte progenitor cells induced according to Experimental Example 1 have the phagocytosis ability, which is a typical characteristic of immune cells, the cell culture medium was treated with green fluorescently labeled latex beads, and whether the beads are introduced into the cytoplasm through phagocytosis was confirmed with a fluorescence microscope, and the results thereof are illustrated in FIG. 4.

[0085] As illustrated in FIG. 4, green beads were introduced into the cytoplasm of the induced monocyte progenitor cells, through which it was confirmed that the induced monocyte progenitor cells had the ability to phagocytize foreign substances.

Experimental Example 4. Confirmation of Immune Response of Induced Monocyte Progenitor Cells

[0086] Monocyte progenitor cells are immune cells and are activated as M1 macrophages that cause an inflammatory response by foreign substances or pathogens or M2 macrophages that have anti-inflammatory action. To confirm the immune response of the monocyte progenitor cells induced according to Experimental Example 1, the induced monocyte progenitor cells were activated by treatment with each of the immune response inducers LPS, IL-4, TGFb1 and IFN-gamma or a combination thereof, and then the morphology of the cell was observed, the expression levels of immune response-related genes were confirmed using the method described in Example 4 through real-time RT-PCR analysis, and the results thereof are illustrated in FIG. 5.

[0087] As illustrated in FIG. 5, it was confirmed that the activated monocyte progenitor cells exhibited the flat amoeba-shaped or rod-shaped cell morphology characteristic of phagocytic cells, and the expression of genes involved in inflammation or anti-inflammatory action was increased.

Experimental Example 5. Confirmation of In-Vivo Immune Disease Treatment Effect of Induced Monocyte Progenitor Cells

[0088] To confirm the in-vivo functional characteristics of the monocyte progenitor cells induced according to Experimental Example 1, the induced monocyte progenitor cells were transplanted into mice of the inflammatory bowel disease animal model produced in Example 5, changes in survival rate and body weight of the mice were analyzed, and the results thereof are illustrated in FIG. 6.

[0089] As illustrated in FIG. 6, it was confirmed that the group transplanted with the induced monocyte progenitor cells exhibited a high survival rate and less weight loss. Disease activity index and maintenance of normal colon length were also improved in the group transplanted with the induced monocyte progenitor cells.