Use of a composition comprising mesenchymal stem cells for the remission of myelofibrosis

Abstract

The use of a composition comprising mesenchymal stem cells (MSCs) in the manufacture of a medicament for alleviating myelofibrosis of interest is provided.

Description

Technical Field

[0001] The present disclosure relates to the use of a composition comprising mesenchymal stem cells for the alleviation of myelofibrosis. Sequence Listing XML The sequence listing submitted simultaneously has a file name of "PE-68332-WO-SEQUENCE LISTING.xml", a creation date of August 24, 2023, a size of 20.4 kilobytes, is part of the specification, and is hereby incorporated by reference in its entirety.

Background Art

[0002] Myelofibrosis is a type of myeloproliferative neoplasm (MPNs), a group of cancers in which abnormal cells proliferate in the bone marrow. Myelofibrosis is often associated with the overactivation of JAK-dependent signaling caused by BCR-ABL1 fusion negativity and JAK2, MPL, CALR mutations. In myelofibrosis, healthy bone marrow is replaced by scar tissue (fibrosis), causing cytopenia, leading to anemia, increased susceptibility to infections, splenomegaly, etc. Myelofibrosis is classified into primary myelofibrosis and secondary myelofibrosis according to the progression of the disease state. Primary myelofibrosis (PMF) is myelofibrosis that occurs without preceding MPNs, while secondary myelofibrosis shows myelofibrosis developing from other MPNs such as essential thrombocythemia (ET) and polycythemia vera (PV), and can be classified into post-ET MF and post-PV MF.

[0003] The only curative treatment for myelofibrosis is allogeneic hematopoietic stem cell transplantation (HSCT). Patients undergo complete or partial resection of the bone marrow (i.e., myeloablative or non-myeloablative HSCT) to remove mutant MPN and stromal cells in the bone marrow, and then hematopoietic stem cells are transplanted from a healthy donor. However, allogeneic HSCT requires human leukocyte antigen matching and has severe complications including graft-versus-host disease (GVHD), so it is only applicable to 5% of patients. So far, the US Food and Drug Administration (FDA) has approved three JAK inhibitors, ruxolitinib (Jakavi), fedratinib (Inrebic), and pacritinib (Vonjo), as therapeutic drugs for moderate or high-risk myelofibrosis. However, these drugs only relieve splenomegaly and some systemic symptoms and do not improve anemia or myelofibrosis.

[0004] In myelofibrosis, fibrosis of bone marrow stromal cells may be induced by TGF-β1, megakaryocytes, or MPN cells. Inflammatory cytokines such as IL-1β, IL-6, and TNF-α released from fibrotic bone marrow stromal cells, megakaryocytes, or MPN cells are greatly involved in the progression of the pathological state of myelofibrosis. By targeting TGF-β1, megakaryocytes, MPN cells, and such inflammatory cytokines, it has been reported that myelofibrosis can be reduced by decreasing bone marrow fibrosis, or indirectly controlling the differentiation and proliferation of megakaryocytes and MPN cells.

[0005] Mesenchymal stem cells (MSCs) have been reported to have immunomodulatory properties when phagocytosed and can thus effectively reduce inflammatory cytokines. In recent years, several studies have reported the use of MSCs in HSCT to control GVHD in patients diagnosed with myelofibrosis. However, the number of cases is limited, and the effectiveness of this approach remains unclear because no improvement in the lifespan of patients after HSCT has been observed.

[0006] Therefore, there is an urgent need to develop new methods for the effective treatment of myelofibrosis.

Summary of the Invention

Problems to be Solved by the Invention

[0007] The object of the present disclosure is to provide the use of a composition for the remission of myelofibrosis that can alleviate at least one drawback of the prior art.

Means for Solving the Problems

[0008] According to the present disclosure, the composition is used for the manufacture of a medicament for alleviating myelofibrosis in a subject.

Brief Description of the Drawings

[0009] Other features and advantages of the present disclosure will become apparent from the following detailed description of the embodiments with reference to the accompanying drawings.

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Mode for Carrying Out the Invention

[0010] Before explaining the present disclosure in more detail, it should be understood that even if any prior art documents are cited herein, the content of such prior art documents does not indicate common general knowledge in the field to which the present invention pertains.

[0011] For the purposes of this specification, the word "comprising" means "including but not limited to", and it should also be understood that the word "include" has a corresponding meaning.

[0012] Unless otherwise defined, all technical and scientific terms used in this specification have the meanings commonly understood by those skilled in the art to which this disclosure pertains. Those skilled in the art can recognize and utilize many methods and materials similar or equivalent to those described herein in the practice of this disclosure. In fact, this disclosure is in no way limited to the described methods and materials.

[0013] To address the current limitations of JAK inhibitors in the treatment of myelofibrosis, the applicant has endeavored to develop improved methods and has discovered that administration of mesenchymal stem cells (MSCs) to a myelofibrosis mouse model can alleviate three major clinical symptoms of myelofibrosis, namely anemia, splenomegaly, and myelofibrosis. Furthermore, in in vitro experiments, it has been shown that MSCs exhibit anti-fibrotic and anti-inflammatory effects in bone marrow stromal cells, megakaryocytes, and MPN cells. Additionally, it has been demonstrated that primed MSCs formed by priming MSCs have higher efficacy against fibrosis of bone marrow stromal cells compared to normal MSCs (i.e., non-primed MSCs).

[0014] Accordingly, the present disclosure provides the use of a composition comprising mesenchymal stem cells (MSCs) in the manufacture of a medicament for alleviating myelofibrosis in a subject.

[0015] Examples of MSCs include, but are not limited to, umbilical cord-derived mesenchymal stem cells (UCMSCs), bone marrow-derived mesenchymal stem cells (BMMSCs), adipose tissue-derived mesenchymal stem cells (AMSCs), dermal-derived mesenchymal stem cells (DMSCs), epidermal-derived mesenchymal stem cells (EMSCs), synovial-derived mesenchymal stem cells (SMMSCs), dental tissue-derived mesenchymal stem cells (dental MSCs), and lung-derived mesenchymal stem cells (LMSCs). In certain embodiments, the MSCs are UCMSCs.

[0016] In certain embodiments, the UCMSCs can be non-primed UCMSCs that have not undergone a priming treatment. In certain embodiments, the UCMSCs can be primed UCMSCs that have undergone a priming treatment. In an exemplary embodiment, the primed UCMSCs are indoleamine pyrrole-2,3-dioxygenase (IDO)-expressing UCMSCs.

[0017] According to the present disclosure, IDO-expressing UCMSCs can be prepared by culturing non-primed UCMSCs in a culture medium supplemented with IFN-γ (interferon γ) for 24 to 72 hours. In an exemplary embodiment, IDO-expressing UCMSCs can be prepared by culturing non-primed UCMSCs in a culture medium supplemented with IFN-γ for 72 hours.

[0018] In certain embodiments, in addition to IFN-γ, a substance was further added to the culture medium. Examples of substances can include, but are not limited to, vitamins, steroids, cytokines, double-stranded RNA, and histone deacetylase (HDAC) inhibitors. In an exemplary embodiment, the vitamin is retinoic acid, the steroids are dexamethasone and budesonide, the cytokine is TNF-α, the double-stranded RNA is polyinosinic acid-polycytidylic acid, and the HDAC inhibitor is valproic acid.

[0019] As used herein, the term "subject" refers to any animal of interest, such as humans, monkeys, cows, sheep, horses, pigs, goats, dogs, cats, mice, rats, etc. In certain embodiments, the subject is a mouse. In certain embodiments, the subject is a human.

[0020] In certain embodiments, the subject has not received myeloablative transplantation.

[0021] As used herein, the term "administer" or "administering" means "introducing, providing, or delivering a predetermined active ingredient to a subject by any suitable route for the purpose of performing an intended function."

[0022] Examples of myelofibrosis can include, but are not limited to, primary myelofibrosis, post-essential thrombocythemia myelofibrosis, and post-polycythemia vera myelofibrosis.

[0023] According to the present disclosure, the pharmaceutical can be formulated into a dosage form suitable for parenteral administration using techniques well known to those skilled in the art.

[0024] According to the present disclosure, for parenteral administration, the pharmaceutical according to the present disclosure can be formulated into an injection such as a sterile aqueous solution, dispersion, or emulsion.

[0025] The pharmaceutical according to the present disclosure can be administered via a parenteral route. The parenteral route includes intraperitoneal injection, intrathoracic injection, intramuscular injection, intravenous injection, internal carotid artery injection, intra-arterial injection, intra-articular injection, intrasynovial injection, intrathecal injection, intracranial injection, intradermal injection, subcutaneous injection, intradermal injection, intralesional injection.

[0026] In certain embodiments, the pharmaceutical is administered by intravenous injection.

[0027] In certain embodiments, a decrease in the level of myelofibrosis or improvement in inflammation is observed in the subject after administration of the pharmaceutical.

[0028] In certain embodiments, improvement in splenomegaly or improvement in anemia is further observed in the subject after administration of the pharmaceutical.

[0029] The dosage and dosing frequency of the pharmaceutical according to the present disclosure can vary depending on the following factors: the severity of the disease or disorder to be treated, the route of administration, and the age, physical condition, and response of the subject to be treated. Generally, the pharmaceutical may be administered once or divided into several doses.

[0030] Hereinafter, examples of the present disclosure will be described. It should be understood that these examples are illustrative and explanatory and should not be construed as limiting the present disclosure.

[0031] Examples General experimental materials 1. Sources and cultures of human bone marrow stromal cells, human erythroleukemia (HEL) cells, megakaryoblast MEG-01 cells, and megakaryoblast SET-2 cells Human bone marrow stromal cells, also known as bone marrow-derived mesenchymal stem cells (BMMSCs), were purchased from ScienCell Research Laboratories, the HEL 92.1.7 cell line and the MEG-01 cell line were obtained from the American Type Culture Collection, and the SET-2 cell line was obtained from the Deutsche Sammlung von Mikroorganismen und Zellkulturen.

[0032] Human bone marrow stromal cells were cultured in α-MEM medium (Gibco) supplemented with 5% human platelet lysate (HPL, purchased from Sexton Biotechnologies). Each of the HEL 92.1.7 cell line and the MEG-01 cell line was cultured in RPMI 1640 medium (Gibco) supplemented with 10% fetal bovine serum (FBS) (Gibco), and the SET-2 cell line was cultured in RPMI 1640 medium supplemented with 20% FBS.

[0033] 2. Source and culture of umbilical cord-derived mesenchymal stem cells (UCMSCs) UCMSCs were isolated from human umbilical cord tissue according to a procedure that modified in part the procedure described in the literature "An improved explant method for the isolation of umbilical cord-derived mesenchymal stem cells and its immunosuppressive properties" by Mori Y. et al. published in Tissue Eng Part C Methods, 2015, Vol. 21, p. 367-372. Briefly, human umbilical cord tissue was immersed in cold phosphate-buffered saline (PBS) and then cut into multiple tissue pieces. The middle part of the umbilical cord tissue pieces was cut to expose Wharton's jelly, and then Wharton's jelly was placed in multiple 10-cm dishes. Next, α-MEM medium supplemented with 10% HPL was immediately added to each dish. The obtained explants were incubated at 37 °C and 5% CO2 for 7 days after inoculation, and then the umbilical cord tissue pieces were removed, and the culture medium (i.e., α-MEM medium supplemented with 5% HPL) was changed every 2 days until the fibroblast-like adherent UCMSCs cells reached 80% - 90% confluence. The UCMSCs migrating from the explants and tissue pieces were collected using TrypLE TM (Gibco) within 10 to 14 days after inoculation.

[0034] Thereafter, the UCMSCs cultures were prepared by seeding UCMSCs at 3000 cells / cm 2 ~5000 cells / cm 2 per culture dish in each culture dish containing α-MEM medium (Gibco) supplemented with 5% HPL (Sexton Biotechnologies). The culture medium was changed every 2 days, and the proliferated UCMSCs were cryopreserved in NutriFreez® D10 cryopreservation medium (manufacturer: Corning).

[0035] 3. Experimental Animals The experimental animals used in the following experiments, namely C57BL / 6 mice, were purchased from BioLASCO. All experimental animals were housed in an animal room equipped with an independent air-conditioning system under laboratory conditions where a 12-hour light period and a 12-hour dark period were repeated alternately, the temperature was maintained at 23°C ± 2°C, and the relative humidity was maintained at 50% ± 10%. The experimental animals were given free access to water and food. All experimental procedures involving the experimental animals complied with the legal regulations of the Animal Protection Law of Taiwan, China, and were carried out in accordance with the guidelines of the Animal Experiment Committee of the Taiwan Agricultural Committee, China.

[0036] General experimental procedures 1. Statistical analysis All the experiments described below were performed at least three times. The experimental data of all test groups were expressed as mean ± standard error of the mean (SEM), and analyzed using Prism 6.0 software (developer: GraphPad Software) with two-sided Student's t-test or one-way analysis of variance followed by Newman-Keuls post hoc test to evaluate the differences between groups. Statistical significance was indicated by p < 0.05.

[0037] Example 1. Preparation of TPO-overexpressing mice and evaluation of the effect of umbilical cord-derived mesenchymal stem cells (UCMSCs) on alleviating myelofibrosis in TPO-overexpressing mice In this example, TPO (thrombopoietin)-overexpressing mice were prepared, the induction of myelofibrosis was verified, and the effect of UCMSCs on alleviating myelofibrosis in TPO-overexpressing mice was evaluated.

[0038] A. Preparation of TPO-overexpressing mice TPO-overexpressing mice were prepared according to the procedure described in the literature "A mouse model of myelofibrosis and osteosclerosis induced by overexpression of thrombopoietin (mpl ligand): recovery of the disease by bone marrow transplantation" by Yan X. Q. et al. in Blood, 1996, Vol. 88, pages 402-409. Briefly, C57BL / 6 mice (6 to 8 weeks old) (n = 6) were intraperitoneally injected with 5-fluorouracil at a dose of 150 mg / kg. Four days after administration, bone marrow tissue was extracted from the mice and incubated with red blood cell (RBC) lysis buffer for 3 minutes. Next, bone marrow cells were seeded into each well of a 12-well plate containing 1 mL of StemSpan SFEM II medium (STEMCELL Technologies Inc.) supplemented with 50 ng / mL of murine stem cell factor (SCF), 50 ng / mL of human TPO, 20 ng / mL of murine IL-3, and 50 ng / mL of murine Flt-3 ligand at a concentration of 5×10 6 cells / well, and immediately infected with a retrovirus containing TPO cDNA (manufacturer: Cell Biolabs, catalog number RV-101) according to the manufacturer's protocol, and a second infection was performed 24 hours after the first infection. Sixteen hours after the second infection, 5×10 6 floating cells were collected as donor cells, and 2×10 5 of these donor cells were transplanted into recipient C57BL / 6 mice that had been pre-irradiated with 17 Gy of gamma rays from cesium 137 by intraorbital injection. Subsequently, the recipient mice were given water containing 1 mM neomycin for 2 weeks to obtain TPO-overexpressing mice.

[0039] Eight weeks after transplantation, to verify the induction of myelofibrosis in TPO-overexpressing mice, the platelet level, RBC level, and hemoglobin level in TPO-overexpressing mice were measured and compared with healthy C57BL / 6 mice (as a control group) to measure the hematopoietic effect. Briefly, blood was collected from the retro-orbital vascular plexus of each mouse, and the platelet count, red blood cell count (RBC), and hemoglobin concentration were measured using a ProCyte Dx hematology analyzer. The results are shown in Figure 1.

[0040] Figure 1 shows the platelet count, RBC count, and hemoglobin concentration of TPO-overexpressing mice and the control group at 8 weeks after transplantation. As shown in Figure 1, compared with the control group, in TPO-overexpressing mice, the platelet count was significantly increased, and the RBC count and hemoglobin concentration were significantly decreased, indicating that the induction of myelofibrosis in TPO-overexpressing mice was successful.

[0041] B. Evaluation of the effect of UCMSCs on alleviating myelofibrosis The cryopreserved UCMSCs were thawed in a 37°C water bath, and the thawed UCMSCs were immersed in α-MEM medium (Gibco) supplemented with 5% HPL (Sexton Biotechnologies), then centrifuged at 350 g for 5 minutes and washed with cold PBS.

[0042] As described in Section A above, the TPO-overexpressing mice that were verified to have myelofibrosis were divided into two groups, namely the pathological control group (PCG) and the experimental group (EG), with n = 3 in each group. Healthy C57BL / 6 mice (n = 3) that had not been transplanted were used as the normal control group (NCG). The mice in the EG group were given 5 × 10 5UCMSCs were administered by intravenous injection via the tail vein at a rate of 100 μL / min, and PBS was administered to the NCG mice. Furthermore, the PCG mice were untreated. At 1, 2, and 3 months after UCMSCs administration, blood was collected from the PCG mice and the EG mice, and the hemoglobin concentration and the number of red blood cells were measured. After blood collection 3 months after UCMSCs administration, the NCG, PCG, and EG mice were sacrificed, and the spleen size was observed and the spleen weight was measured. Furthermore, the bone marrow of the PCG and EG mice was subjected to identification of myelofibrosis using hematoxylin-eosin (H&E) staining and periodic acid Schiff (PAS) staining that can stain carbohydrates in various collagens, and the procedure described in the literature "Periodic acid Schiff (PAS) reaction and plastination in whole body slices. A new method for identifying fascial tissue structure" published by Steinke H. et al. in Ann Anat, 2018, Vol.216, p.29-35. Furthermore, the grading of myelofibrosis was performed according to the procedure described in the literature "European consensus on grading of myelofibrosis and assessment of cellularity" published by Thiele J. et al. in Haematologica, 2005, Vol.90, p.1128-1132. The results are shown in Figs. 2(a)-(f).

[0043] Fig. 2(a) shows the hemoglobin concentration (left panel) and the number of RBCs (right panel) in the mice of each group at 0 months (i.e., 8 weeks after transplantation), 1 month, 2 months, and 3 months after UCMSCs administration. As shown in Fig. 2(a), from 0 months to 3 months after UCMSCs administration, the hemoglobin concentration and the number of red blood cells gradually decreased in the mice of the pathological control group, while in the mice of the experimental group, the hemoglobin concentration and the number of red blood cells increased 2 months after UCMSCs administration, indicating that anemia was improved in the TPO overexpression mice after UCMSCs administration.

[0044] Figures 2(b) and (c) show the size and weight of the spleens of the mice in each group 3 months after administration of UCMSCs. As shown in Figures 2(b) and (c), the spleens of the mice in the pathological control group were larger in size and heavier in weight compared to those of the mice in the normal control group, while the spleens of the mice in the experimental group were decreased in size and weight compared to those in the pathological control group, indicating that splenomegaly was improved in TPO overexpressing mice after treatment with UCMSCs.

[0045] Figures 2(d) and (e) represent the bone marrow of the mice in each group 3 months after administration of UCMSCs stained with hematoxylin-eosin (H&E) and periodic acid Schiff (PAS). As shown in Figures 2(d) and (e), migration of atypical cells was observed in the pathological control group (indicated by the circled area), but not in the experimental group. On the other hand, the number of (PAS + ) cells (indicated by arrows) was higher in the pathological control group than in the experimental group.

[0046] Figure 2(f) shows the relative percentage of the PAS + area (left panel) and the relative percentage of the PAS + intensity (right panel) of the bone marrow of the mice in each group 3 months after administration of UCMSCs. As shown in Figure 2(f), compared with the pathological control group, the relative percentage of the PAS + area and the relative percentage of the PAS + intensity in the experimental group decreased to 61.34 ± 4.93% and 61.45 ± 4.97%, respectively, indicating that the level of bone marrow fibrosis decreased in TPO overexpressing mice after treatment with UCMSCs.

[0047] These results demonstrated that in the mouse model of myelofibrosis, UCMSCs could alleviate the three major clinical symptoms of myelofibrosis, namely anemia, splenomegaly, and bone marrow fibrosis.

[0048] Example 2. Evaluation of the effect of UCMSCs in alleviating myelofibrosis induced by various factors In this example, a cell model of myelofibrosis was established using human bone marrow stromal cells and / or myeloproliferative neoplasm (MPN) cells, and then the effect of UCMSCs on such a cell model was determined.

[0049] A. Effect of UCMSCs on TGF-β1-induced fibrosis and inflammation in human bone marrow stromal cells Since TGF-β1 has been reported to mediate bone marrow fibrosis in myelofibrosis, in this experiment, human bone marrow stromal cells were incubated with TGF-β1 and subsequently treated with UCMSCs, and the expression levels of gene markers of fibrosis and inflammation were measured.

[0050] First, human bone marrow stromal cells were divided into six groups, specifically, a normal control group (NCG), a pathological control group (PCG), a comparison group (CG), and three experimental groups, namely experimental groups 1 to 3 (EG1 to EG3). The number of cells in each group was 4×10 4 cells. Then, the human bone marrow stromal cells in the PCG, EG1 to EG3 were incubated with 5 ng / mL of TGF-β1 for 72 hours to induce a fibrotic and inflammatory response, while the human bone marrow stromal cells in the NCG and CG were not treated with TGF-β1. Subsequently, the human bone marrow stromal cells in the CG, EG1, EG2, and EG3 were treated with UCMSCs for 72 hours at a cell number ratio of UCMSCs to human bone marrow stromal cells of 1:1, 1:4, 1:2, and 1:1, respectively, while the human bone marrow stromal cells in the NCG and CG were not treated. Subsequently, the relative mRNA expression levels of fibrotic marker genes, namely collagen type I alpha 1 chain (COL1A1), fibronectin 1 (FN1), and actin alpha 2 (ACTA2), and the relative mRNA expression levels of inflammatory marker genes, namely IL-1β, TNF-α, TGF-β1, and IL-6, were measured in the human bone marrow stromal cells of each group. In this experiment, the relative mRNA expression level of IL-6 in the human bone marrow stromal cells of each group was not measured.

[0051] Specifically, after removing UCMSCs, human bone marrow stromal cells in each group were subjected to total RNA extraction using the RNeasy Mini Kit (QIAGEN) according to the manufacturer's instructions. The RNA obtained from each group was used as a template for synthesizing cDNA by reverse transcription polymerase chain reaction (RT-PCR) using the GScript First-Strand Synthesis Kit (GeneDirex) and FastStart Essential DNA Green Master (Roche). Using the obtained cDNA as a DNA template, specific designed primer pairs for six marker genes shown in Table 1, namely the COL1A1 gene, FN1 gene, ACTA2 gene, IL-1β gene, TNF-α gene, and TGF-β1 gene, and the reaction conditions shown in Table 2, SYBR-Green I fluorescence-based quantitative real-time PCR was performed on a LightCycler® 96 system (Roche). The peptidylprolyl isomerase A (PPIA) gene was used as an endogenous control in the quantitative analysis of real-time PCR for normalizing gene expression data.

[0052]

Table 1

[0053]

Table 2

[0054] The fluorescence intensity of the obtained PCR was measured, and the cycle threshold (Ct) values of each of the COL1A1 gene, FN1 gene, ACTA2 gene, IL-1β gene, TNF-α gene, and TGF-β1 gene were calculated. Quantitative real-time PCR data were analyzed using the comparative Ct method. Briefly, the Ct value of each marker gene (i.e., the COL1A1 gene, FN1 gene, ACTA2 gene, IL-1β gene, TNF-α gene, and TGF-β1 gene) in each group was normalized by the Ct value of the PPIA gene, and the relative mRNA expression level of each marker gene was further calculated using the following formula (I).

[0055] A = 2 -(B-C) (I) Where A = the relative mRNA expression level of each marker gene B = the normalized Ct value of each marker gene in each group C = the normalized Ct value of each marker gene in the normal control group

[0056] The results are shown in Figure 3.

[0057] Figure 3 shows the relative mRNA expression levels of six marker genes, namely, (a) COL1A1, (b) FN1, (c) ACTA2, (d) IL-1β, (e) TNF-α, and (f) TGF-β1, in human bone marrow stromal cells of each group. As shown in Figures 3(a) to 3(c), the respective relative mRNA expression levels of COL1A1, FN1, and ACTA2 measured in the pathological control group were significantly higher than the levels measured in the normal control group, indicating that fibrosis was induced in human bone marrow stromal cells after incubation with TGF-β1. Furthermore, the relative mRNA expression level of COL1A1 measured in experimental groups 2 and 3 was significantly decreased compared with the level measured in the pathological control group, and the respective relative mRNA expression levels of FN1 and ACTA2 measured in experimental groups 1 to 3 were significantly decreased compared with the levels measured in the pathological control group, indicating that the fibrosis of human bone marrow stromal cells was significantly improved by treatment with UCMSCs. In contrast, as shown in Figures 3(d) to 3(f), there was no significant difference in the respective relative mRNA expression levels of IL-1β, TNF-α, and TGF-β1 measured in the pathological control group and experimental groups 1 to 3 compared with the normal control group, suggesting that inflammation was not significantly induced in human bone marrow stromal cells after incubation with TGF-β1 and that UCMSCs did not exert an anti-inflammatory effect on human bone marrow stromal cells.

[0058] B. Effects of UCMSCs on TGF-β1-induced and MPN cell-induced fibrosis and inflammation in human bone marrow stromal cells JAK2 V617F Since it has been reported that MPN mononuclear cells with mutations induce inflammation in human bone marrow stromal cells, in this experiment, in order to mimic the post-PV MF cell model, JAK2 V617F positive MPN cell line HEL92.1.7 cells were combined with human bone marrow stromal cells and incubated with TGF-β1, and then treated with UCMSCs, and the expression levels of gene markers of fibrosis and inflammation were measured.

[0059] Briefly, human bone marrow stromal cells were divided into six groups, specifically, a normal control group (NCG), three pathological control groups (PCG1 - PCG3), and two experimental groups, namely experimental groups 1 - 2 (EG1 and EG2), with the number of cells in each group being 4×10 4 cells. Next, the human bone marrow stromal cells of PCG3, EG1, and EG2 were co-cultured with 1×10 5 HEL 92.1.7 cells for 72 hours in the presence of 5 ng / mL TGF-β1, the human bone marrow stromal cells of PCG1 were incubated with 5 ng / mL TGF-β1 for 72 hours, the human bone marrow stromal cells of PCG2 were co-cultured with 1×10 5 HEL 92.1.7 cells for 72 hours, and no treatment was given to the human bone marrow stromal cells of NCG. After separating the HEL 92.1.7 cells from the human bone marrow stromal cells, the human bone marrow stromal cells of EG1 and EG2 were treated with UCMSCs for 72 hours such that the ratios of the number of UCMSCs to human bone marrow stromal cells were 1:4 and 1:2 respectively, while no treatment was given to the human bone marrow stromal cells of NCG and PCG1 - PCG3. Then, after removing the UCMSCs, total RNA extraction, cDNA synthesis, real-time PCR, normalization of gene expression data, and calculation of the relative mRNA expression levels of COL1A1, FN1, ACTA2, IL-1β, TNF-α, and TGF-β1 were performed on the human bone marrow stromal cells of each group using the procedures and conditions described in Section A above. The results are shown in Figure 4.

[0060] Figure 4 shows the relative expression levels of (a) COL1A1, (b) FN1, (c) ACTA2, (d) IL-1β, (e) TNF-α, and (f) TGF-β1 in human bone marrow stromal cells of each group. As shown in Figure 4, the respective relative expression levels of COL1A1, FN1, ACTA2, IL-1β, TNF-α, and TGF-β1 measured in pathological control group 3 were significantly higher than the levels measured in the normal control group, indicating that fibrosis was induced in human bone marrow stromal cells after co-culture with HEL92.1.7 cells in the presence of TGF-β1. Furthermore, the relative mRNA expression level of COL1A1 measured in experimental group 2 was significantly decreased compared with the level measured in pathological control group 3, and the respective relative mRNA expression levels of FN1, ACTA2, IL-1β, and TNF-α measured in experimental groups 1 and 2 were significantly decreased compared with the levels measured in pathological control group 3, and also, the relative mRNA expression level of TGF-β1 measured in experimental group 2 was significantly decreased compared with the level measured in pathological control group 3, indicating that UCMSCs exert anti-fibrotic and anti-inflammatory effects on human bone marrow stromal cells.

[0061] C. Effects of UCMSCs on megakaryocyte-induced fibrosis and inflammation in human bone marrow stromal cells Megakaryocytes have been reported to induce bone marrow fibrosis and inflammation in post-ET MF and PMF, and MEG-01 cells have been reported to enhance fibrosis in human bone marrow stromal cells. Therefore, in this experiment, human bone marrow stromal cells were co-cultured with MEG-01 cells and then treated with UCMSCs to measure the expression levels of gene markers of fibrosis and inflammation.

[0062] In short, human bone marrow stromal cells were divided into three groups, specifically, a normal control group (NCG), a pathological control group (PCG), and an experimental group (EG), and the number of cells in each group was 4×10 4 cells. Next, the human bone marrow stromal cells of PCG and EG were 1×10 5The MEG-01 cells were co-cultured with the NCG human bone marrow stromal cells for 48 hours without any treatment. After separating the MEG-01 cells from the human bone marrow stromal cells, the EG human bone marrow stromal cells were treated with UCMSCs for 72 hours such that the cell number ratio of UCMSCs to human bone marrow stromal cells was 1:1, while the NCG and PCG human bone marrow stromal cells were not treated at all. Subsequently, after removing the UCMSCs, total RNA extraction, cDNA synthesis, real-time PCR, normalization of gene expression data, and calculation of the relative mRNA expression levels of COL1A1, FN1, ACTA2, IL-1β, and IL-6 were performed on the human bone marrow stromal cells of each group using the procedures and conditions described in Section A above. The results are shown in Figure 5.

[0063] Figure 5 shows the relative expression levels of (a) COL1A1, (b) FN1, (c) ACTA2, (d) IL-1β, and (e) IL-6 in the human bone marrow stromal cells of each group. As shown in Figure 5, the respective relative expression levels of COL1A1, FN1, ACTA2, IL-1β, and IL-6 measured in the pathological control group were higher than the levels measured in the normal control group, indicating that fibrosis and inflammation were induced in the human bone marrow stromal cells after co-culture with MEG-01 cells. Furthermore, the respective relative mRNA expression levels of COL1A1, FN1, ACTA2, IL-1β, and IL-6 measured in the experimental group were decreased compared to the levels measured in the pathological control group, indicating that UCMSCs exerted anti-fibrotic and anti-inflammatory effects on the human bone marrow stromal cells.

[0064] IL-1β has been reported to mediate myelofibrosis in SET-2 cells, which are megakaryocytes with JAK2 V617F mutations. Therefore, human bone marrow stromal cells were co-cultured with SET-2 cells in the presence of IL-1β and subsequently treated with UCMSCs to measure the expression levels of gene markers of fibrosis and inflammation.

[0065] Briefly, human bone marrow stromal cells were divided into six groups, specifically, a normal control group (NCG), two pathological control groups (PCG1 and PCG2), and three experimental groups, namely experimental groups 1 to 3 (EG1 to EG3), with the number of cells in each group being 4×10 4 cells. Next, each of the human bone marrow stromal cells in PCG2 and EG1 to EG3 was co-cultured with 1×10 5 SET-2 cells (note: SET-2 cells were first treated with 30 ng / mL of IL-1β for 24 hours and then IL-1β was removed) for 72 hours. The human bone marrow stromal cells in PCG1 were co-cultured with 1×10 5 SET-2 cells for 72 hours, and no treatment was given to the human bone marrow stromal cells in NCG. After separating the SET-2 cells from the human bone marrow stromal cells, the human bone marrow stromal cells in EG1 to EG3 were treated with UCMSCs for 72 hours such that the ratio of the number of UCMSCs to the number of human bone marrow stromal cells was 1:10, 1:4, and 1:2, respectively, while the human bone marrow stromal cells in NCG, PCG1, and PCG2 were not treated. Subsequently, after removing the UCMSCs, total RNA extraction, cDNA synthesis, real-time PCR, normalization of gene expression data, and calculation of the relative mRNA expression levels of COL1A1, FN1, ACTA2, IL-1β, and IL-6 were performed on the human bone marrow stromal cells in each group using the procedures and conditions described in Section A above. The results are shown in Figure 6.

[0066] Figure 6 shows the relative expression levels of (a) COL1A1, (b) FN1, (c) ACTA2, (d) IL-1β, and (e) IL-6 in human bone marrow stromal cells of each group. As shown in Figure 6, the respective relative expression levels of COL1A1, FN1, IL-1β, and IL-6 measured in the pathological control group 2 were significantly higher than the levels measured in the normal control group, indicating that fibrosis and inflammation were induced in human bone marrow stromal cells after co-culture with IL-1β-pretreated SET-2 cells. Furthermore, the relative mRNA expression level of COL1A1 measured in experimental group 3 was significantly decreased compared with the level measured in the pathological control group, the relative mRNA expression levels of FN1 measured in experimental groups 1 to 3 were significantly decreased compared with the level measured in the pathological control group, and the relative mRNA expression level of IL-6 measured in experimental group 3 was significantly decreased compared with that measured in the pathological control group, indicating that UCMSCs exert anti-fibrotic and anti-inflammatory effects on human bone marrow stromal cells.

[0067] D. Effect of UCMSCs on human bone marrow stromal cell-induced inflammation in MPN cells Human bone marrow stromal cells have been reported to induce the release of inflammatory cytokines from megakaryocytes in post-ET MF and PMF, which is highly involved in the pathological progression of myelofibrosis. In this experiment, an MPN cell line, namely MEG-01 cells, was incubated with human bone marrow stromal cells, and then treated with UCMSCs to measure the expression levels of inflammatory gene markers.

[0068] In short, MEG-01 cells were divided into three groups, specifically, a normal control group (NCG), a pathological control group (PCG), and an experimental group (EG), and the number of MEG-01 cells in each group was 1×10 5 cells. Next, the MEG-01 cells in the PCG and EG were co-cultured with 4×10 4 human bone marrow stromal cells for 48 hours to induce an inflammatory response, while the MEG-01 cells in the NCG were not treated with anything. After separating the human bone marrow stromal cells from the MEG-01, the MEG-01 in the EG was treated with 4×10 4UCMSCs were treated for 72 hours at a ratio of the number of UCMSCs to MEG-01 cells of 1:1, while the MEG-01 cells in the NCG and PCG were not treated at all. After removing the UCMSCs, total RNA extraction, cDNA synthesis, real-time PCR, normalization of gene expression data, and calculation of the relative mRNA expression levels of IL-1β, IL-6, TNF-α, and TGF-β1 were performed on the MEG-01 cells in each group using the procedures and conditions described in Section A above. The results are shown in Figure 7.

[0069] Figure 7 shows the relative expression levels of (a) IL-1β, (b) IL-6, (c) TNF-α, and (d) TGF-β1 in MEG-01 cells of each group. As shown in Figure 7, the respective relative expression levels of IL-1β, IL-6, TNF-α, and TGF-β1 measured in the pathological control group were higher than the levels measured in the normal control group, indicating that inflammation was induced in MEG-01 cells after co-culture with human bone marrow stromal cells. Furthermore, the respective relative mRNA expression levels of IL-1β, IL-6, TNF-α, and TGF-β1 measured in the experimental group decreased compared to the levels measured in the pathological control group, indicating that UCMSCs exert an anti-inflammatory effect on MEG-01 cells.

[0070] Effect of UCMSCs on cytokine-induced inflammation in E.MPN cells Since IL-1β has been reported to mediate myelofibrosis in the post-PV cell model, in this experiment, another MPN cell line, namely HEL 92.1.7 cells, was incubated with IL-1β and subsequently treated with UCMSCs to measure the expression levels of inflammatory gene markers.

[0071] In short, HEL 92.1.7 cells were divided into four groups, specifically, a normal control group (NCG), a pathological control group (PCG), and two experimental groups, namely experimental group 1 and 2 (EG1 and EG2), and the number of HEL 92.1.7 cells in each group was 2.5×10 4There were [number] of them. Next, HEL 92.1.7 cells of PCG, EG1, and EG2 were incubated with 30 ng / mL of IL-1β for 72 hours to induce an inflammatory response, while no treatment was given to HEL 92.1.7 cells of NCG. Then, HEL 92.1.7 cells of EG1 and EG2 were treated with UCMSCs for 48 hours such that the ratio of the cell numbers of UCMSCs to HEL 92.1.7 was 1:4 and 1:2 respectively, while HEL 92.1.7 cells of NCG were not treated at all. After removing UCMSCs, total RNA extraction, cDNA synthesis, real-time PCR, normalization of gene expression data, and calculation of the relative mRNA expression levels of IL-6 and TNF-α were performed on HEL 92.1.7 cells of each group using the procedures and conditions described in Section A above. The results are shown in Figure 8.

[0072] Figure 8 shows the relative expression levels of (a) IL-6 and (b) TNF-α in HEL 92.1.7 cells of each group. As shown in Figure 8, the respective relative expression levels of IL-6 and TNF-α measured in the pathological control group were higher than the values measured in the normal control group, indicating that inflammation was induced in HEL 92.1.7 cells after incubation with IL-β. Furthermore, the respective relative mRNA expression levels of IL-6 and TNF-α measured in experimental groups 1 and 2 decreased compared to the levels measured in the pathological control group, indicating that UCMSCs exerted an anti-inflammatory effect on HEL 92.1.7 cells.

[0073] F. Effects of MSCs from different sources on TGF-β1-induced fibrosis in human bone marrow stromal cells To verify the anti-fibrotic effect of MSCs, human bone marrow stromal cells induced to fibrosis by TGF-β1 were treated with MSCs from various sources including umbilical cord-derived MSCs (UCMSCs), adipose tissue-derived MSCs (AMSCs), and lung-derived MSCs (LMSCs), and the expression levels of gene markers of fibrosis were measured.

[0074] Briefly, human bone marrow stromal cells were divided into five groups, specifically, a normal control group (NCG), a pathological control group (PCG), and three experimental groups, namely experimental groups 1 to 3 (EG1 to EG3). The number of human bone marrow stromal cells in each group was 4×10 4 cells. Next, the human bone marrow stromal cells in PCG and EG1 to EG3 were incubated with 5 ng / mL of TGF-β1 for 72 hours, while the human bone marrow stromal cells in NCG were not treated with anything. Thereafter, the human bone marrow stromal cells in EG1 to EG3 were treated with 2×10 4 cells of UCMSCs, 2×10 4 cells of AMSCs, and 2×10 4 cells of LMSCs for 72 hours, and the human bone marrow stromal cells in NCG and PCG were not treated with anything. Thereafter, after removing UCMSCs, total RNA extraction, cDNA synthesis, real-time PCR, normalization of gene expression data, and calculation of the relative mRNA expression levels of COL1A1 and FN1 were performed on the human bone marrow stromal cells in each group using the procedures and conditions described in Section A above. The results are shown in Figure 9.

[0075] Figure 9 shows the relative expression levels of (a) COL1A1 and (b) FN1 in human bone marrow stromal cells of each group. As shown in Figure 9, the respective relative expression levels of COL1A1 and FN1 measured in the pathological control group were significantly higher than the levels measured in the normal control group, indicating that fibrosis was induced in human bone marrow stromal cells after incubation with TGF-β1. Furthermore, the respective relative mRNA expression levels of COL1A1 and FN1 measured in experimental groups 1 to 3 were decreased compared to the levels measured in the pathological control group, indicating that UCMSCs, AMSCs, and LMSCs can each exert an anti-fibrotic effect on human bone marrow stromal cells.

[0076] G. Effects of MSCs from different sources on MPN cell-induced fibrosis in human bone marrow stromal cells To further verify the anti-fibrotic effect of MSCs, human bone marrow stromal cells containing MPN cells with induced fibrosis in the presence of IL-1β were treated with MSCs from different sources including UCMSCs, AMSCs, and LMSCs, and the expression levels of gene markers of fibrosis were measured.

[0077] In short, human bone marrow stromal cells were divided into five groups, specifically, a normal control group (NCG), a pathological control group (PCG), and three experimental groups, namely experimental groups 1 - 3 (EG1 - EG3). The number of human bone marrow stromal cells in each group was 4×10 4 cells. Next, each of the human bone marrow stromal cells in PCG and EG1 - EG3 was co-cultured with 1×10 5 SET-2 cells (Note: SET-2 cells were first treated with 30 ng / mL of IL-1β for 24 hours and then IL-1β was removed) for 72 hours, while the human bone marrow stromal cells in NCG were not treated with anything. After separating the SET-2 cells from the human bone marrow stromal cells, the human bone marrow stromal cells in EG1 - EG3 were each treated with 2×10 4 UCMSCs, 2×10 4 AMSCs, 2×10 4 LMSCs for 72 hours, while the human bone marrow stromal cells in NCG and PCG were not treated with anything. Then, after removing UCMSCs, AMSCs, and LMSCs respectively, for the human bone marrow stromal cells in each group, total RNA extraction, cDNA synthesis, real-time PCR, normalization of gene expression data, and calculation of the relative mRNA expression levels of COL1A1 and FN1 were performed using the procedures and conditions described in Section A above. The results are shown in Figure 10.

[0078] Figure 10 shows the relative expression levels of (a) COL1A1 and (b) FN1 in human bone marrow stromal cells of each group. As shown in Figure 10, the respective relative expression levels of COL1A1 and FN1 measured in the pathological control group were significantly higher than the levels measured in the normal control group, indicating that fibrosis was induced in human bone marrow stromal cells after co-culture with SET-2 cells pretreated with IL-1β. Furthermore, the relative mRNA expression levels of COL1A1 and FN1 measured in Experimental Groups 1 to 3 were decreased compared to the levels measured in the pathological control group, indicating that UCMSCs, AMSCs, and LMSCs each can exert an anti-fibrotic effect on human bone marrow stromal cells.

[0079] Example 3. Evaluation of the Effect of Primed UCMSCs on Alleviating Myelofibrosis In this example, primed UCMSCs, that is, indoleamine pyrrole-2,3-dioxygenase-positive (IDO)-expressing UCMSCs, were prepared and then evaluated using the cell model of myelofibrosis as described in Example 2 above to measure the effectiveness of primed UCMSCs against fibrosis.

[0080] A. Preparation of Primed UCMSCs The UCMSC cultures described in Item 2 of General Experimental Materials were divided into six groups, specifically, a control group (CG) and five experimental groups, namely Experimental Groups 1 to 5 (EG1 to EG5). The number of UCMSCs in each group was 5.8×10 5They are cells. Next, UCMSCs of EG1 - EG5 were each primed by treatment with different concentrations of IFN - γ at 50, 100, 200, 250, and 500 U / mL for 72 hours, while the UCMSCs of the CG were not subjected to any treatment. For the identification of IDO - expressing UCMSCs, the cells of each group were stained with a phycoerythrin - conjugated IDO monoclonal antibody (manufacturer: Thermo Fisher Scientific, catalog number: 12 - 9477 - 42). On the other hand, a phycoerythrin - conjugated mouse IgG1 kappa isotype control (manufacturer: Thermo Fisher Scientific, catalog number: 12 - 4714 - 82) was used as a control antibody, and the stained cells were subjected to flow cytometry using a CytoFLEX flow cytometer (Beckman Coulter) to measure the percentage of IDO - expressing UCMSCs in each group. The results are shown in Figure 11.

[0081] Figure 11 shows the percentage of IDO - expressing UCMSCs in each group after treatment with different concentrations of IFN - γ for 72 hours. As shown in Figure 11, the percentage of IDO - expressing UCMSCs, namely primed UCMSCs, measured in each of experimental groups 1 - 5 was higher compared to the control group, and such an increase was dependent on the concentration of IFN - γ.

[0082] From the above - mentioned results, since it was shown that the percentage of IDO - expressing UCMSCs was higher in UCMSCs treated with 250 U / mL and 500 U / mL of IFN - γ, in the next experiment, UCMSCs were treated with these concentrations of IFN - γ for different periods, and the percentage of IDO - expressing UCMSCs was measured.

[0083] Briefly, the UCMSCs cultures described in item 2 of the general experimental materials were divided into 9 groups, specifically, 3 control groups, namely, control groups 1-3 (CG1-CG3), and 6 experimental groups, namely, experimental groups 1-1 (EG1-1), experimental groups 1-2 (EG1-2), experimental groups 2-1 (EG2-1), experimental groups 2-2 (EG2-2), experimental groups 3-1 (EG3-1) and experimental groups 3-2 (EG3-2). The number of UCMSCs in each group was 5.8×10 5 cells. Next, the UCMSCs of EG1-1, EG2-1, and EG3-1 were each primed by treatment with 250 U / mL of IFN-γ for 24 hours, 48 hours, and 72 hours, and the UCMSCs of EG1-2, EG2-2, and EG3-2 were each treated with 500 U / mL of IFN-γ for 24 hours, 48 hours, and 72 hours. The UCMSCs of CG1, CG2, and CG3 were not treated and were left for 24 hours, 48 hours, and 72 hours, respectively. Then, the primed UCMSCs obtained in each group were subjected to flow cytometry according to the above-mentioned procedure, and the percentage of IDO-expressing UCMSCs in each group was measured. The results are shown in Figure 12.

[0084] Figure 12 shows the percentage of IDO-expressing UCMSCs in each group after treatment with IFN-γ at different concentrations for different periods. As shown in Figure 12, the percentages of IDO-expressing UCMSCs, i.e., primed UCMSCs, measured in experimental groups 1-1 and 1-2 were higher compared to control group 1, the percentages of IDO-expressing UCMSCs measured in experimental groups 2-1 and 2-2 were higher compared to control group 2, and the percentages of IDO-expressing UCMSCs measured in experimental groups 3-1 and 3-2 were higher compared to control group 3. Also, the percentage of IDO-expressing UCMSCs measured in experimental group 3-1 was higher compared to experimental groups 2-1 and 1-1, and the percentage of IDO-expressing UCMSCs measured in experimental group 3-2 was higher compared to experimental groups 2-2 and 1-2. These results suggest that the increase in the percentage of IDO-expressing UCMSCs depends on the concentration of IFN-γ and the treatment period.

[0085] The following experiments were conducted to determine whether the addition of supplements selected from vitamins, steroids, cytokines, double-stranded RNA, and histone deacetylase (HDAC) inhibitors enhances the effect of IFN-γ in the priming of UCMSCs.

[0086] Briefly, the UCMSCs cultures described in item 2 of the general experimental materials were divided into 8 groups, specifically, a normal control group (NCG), a comparison group (CG), and 6 experimental groups, namely experimental groups 1 to 6 (EG1 to EG6). The number of UCMSCs in each group was 5.8×10 5 cells. Next, the UCMSCs in each group were primed. The UCMSCs in the CG were treated with 250 U / m of IFN-γ for 72 hours, and the UCMSCs in EG1 to EG6 were treated with 250 U / mL of IFN-γ for 72 hours in combination with 1 μM of retinoic acid, 1 μg / mL of dexamethasone, 10 ng / mL of TNF-α, 25 μg / mL of polyinosinic acid-polycytidylic acid, 0.5 mM of valproic acid, or 1 μM of budesonide, respectively. The UCMSCs in the NCG were left untreated for 72 hours. Subsequently, for the primed UCMSCs obtained in each group, total RNA extraction, cDNA synthesis, real-time PCR using the IDO gene-specific primer pairs shown in Table 3 below, normalization of gene expression data, and calculation of the relative mRNA expression level of the IDO gene were performed using the procedures and conditions described in Section A of Example 2. The results are shown in Figure 13.

[0087]

Table 3

[0088] Figure 13 shows the relative mRNA expression levels of the IDO gene in each group after treatment with IFN-γ in combination with each supplement. As shown in Figure 13, the relative mRNA expression levels of the IDO gene measured in each of Experimental Groups 1-6 were higher than the levels measured in the control group, and the number of primed UCMSCs measured after treating UCMSCs with IFN-γ in combination with any one of retinoic acid, dexamethasone, TNF-α, polyinosinic acid-polycytidylic acid, valproic acid, and budesonide was shown to be even more than the number of primed UCMSCs measured after treating UCMSCs with IFN-γ alone. These results suggest that each of retinoic acid, dexamethasone, TNF-α, polyinosinic acid-polycytidylic acid, valproic acid, and budesonide can enhance the effect of IFN-γ in priming UCMSCs.

[0089] From the above results, since it was demonstrated that the relative mRNA expression level of the IDO gene increased the most in UCMSCs treated with IFN-γ in combination with budesonide, the following experiment was conducted to examine the effect on the percentage of IDO-expressing UCMSCs when UCMSCs were treated with IFN-γ and budesonide for different periods.

[0090] In short, the UCMSCs culture described in Item 2 of the general experimental materials was divided into 8 groups, specifically, 3 control groups, namely Control Groups 1-3 (CG1-CG3), and 5 experimental groups, namely Experimental Group 1-1 (EG1-1), Experimental Group 1-2 (EG1-2), Experimental Group 2-1 (EG2-1), Experimental Group 2-2 (EG2-2), and Experimental Group 3-2 (EG3-2). The number of UCMSCs in each group was 5.8×10 5 cells. The priming process was carried out by treating UCMSCs with 250 U / mL of IFN-γ and / or 1 μM of budesonide for a total of 72 hours. The treatment periods with IFN-γ and / or budesonide for UCMSCs in each group are shown in Table 4 below.

[0091]

Table 4

[0092] Subsequently, the primed UCMSCs obtained in each group were subjected to flow cytometry according to the aforementioned procedure, and the relative percentage of IDO-expressing UCMSCs in each group was measured. The results are shown in Figure 14.

[0093] Figure 14 shows the relative percentages of IDO-expressing UCMSCs in each group after IFN-γ treatment for different periods in combination with budesonide for different periods. As shown in Figure 14, the relative percentages of IDO-expressing UCMSCs, namely primed UCMSCs, measured in experimental groups 1-1, 1-2, 2-1, 2-2, and 3-2 were higher compared to the percentages measured in control groups 1-3, indicating that budesonide can enhance the effect of IFN-γ in priming UCMSCs. Furthermore, the percentage of IDO-expressing UCMSCs measured in experimental group 1-2 was higher compared to the percentage measured in experimental group 1-1, and the percentage of IDO-expressing UCMSCs measured in experimental group 2-2 was higher compared to the percentage measured in experimental group 2-1, demonstrating that the effect of IFN-γ in priming UCMSCs is further enhanced by relatively long-term budesonide treatment.

[0094] B. Effect of Primed UCMSCs on TGF-β1-Induced and MPN Cell-Induced Fibrosis in Human Bone Marrow Stromal Cells To measure the effect of primed UCMSCs and normal (i.e., non-primed) UCMSCs in a post-PV MF cell model, in this experiment, human bone marrow stromal cells were co-cultured with HEL92.1.7 cells in the presence of TGF-β1 and then treated with normal UCMSCs or primed UCMSCs, and the expression levels of fibrosis gene markers in human bone marrow stromal cells were measured.

[0095] Briefly, human bone marrow stromal cells were divided into six groups, specifically, a normal control group (NCG), a pathological control group (PCG), three comparison groups, namely comparison groups 1 - 3 (CG1 - CG3), and an experimental group (EG). The number of human bone marrow stromal cells in each group was 4×10 4 cells. Next, the human bone marrow stromal cells of PCG, CG1, CG2, CG3, and EG were co - cultured with 4×10 4 HEL 92.1.7 cells in the presence of 5 ng / mL TGF - β1 for 72 hours to induce fibrosis, while no treatment was given to the human bone marrow stromal cells of NCG. After separating HEL 92.1.7 cells from the human bone marrow stromal cells, the human bone marrow stromal cells of CG1 - CG3 were treated with normal UCMSCs for 72 hours so that the cell number ratio of normal UCMSCs to human bone marrow stromal cells was 1:10, 1:4, and 1:2 respectively, and the human bone marrow stromal cells of EG were treated with priming UCMSCs for 72 hours so that the cell number ratio of priming UCMSCs to human bone marrow stromal cells was 1:10, while no treatment was given to the human bone marrow stromal cells of NCG and PCG. Then, after removing normal UCMSCs or priming UCMSCs, total RNA extraction, cDNA synthesis, real - time PCR, normalization of gene expression data, and calculation of the relative mRNA expression levels of COL1A1, FN1, and ACTA2 were performed on the human bone marrow stromal cells of each group using the procedures and conditions described in Section A of Example 2 above. The results are shown in Figure 15.

[0096] Figure 15 shows the relative expression levels of (a) COL1A1, (b) FN1, and (c) ACTA2 in human bone marrow stromal cells of each group. As shown in Figure 15, the respective relative expression levels of COL1A1, FN1, and ACTA2 measured in the pathological control group were significantly higher than the levels measured in the normal control group, indicating that fibrosis was induced in human bone marrow stromal cells after co-culture with HEL92.1.7 cells in the presence of TGF-β1. Furthermore, the relative mRNA expression level of COL1A1 measured in Comparative Groups 1-3 was significantly decreased compared to the level measured in the pathological control group, and the relative mRNA expression level of FN1 measured in Comparative Group 3 was significantly decreased compared to the level measured in the pathological control group, indicating that the fibrosis of human bone marrow stromal cells was significantly improved by treatment with normal UCMSCs. Additionally, the respective relative mRNA expression levels of COL1A1, FN1, and ACTA2 measured in the experimental group were significantly decreased compared to the levels measured in Comparative Group 1, indicating that primed UCMSCs enhance the effect on fibrosis in human bone marrow stromal cells compared to normal UCMSCs.

[0097] C. Effect of Primed UCMSCs on Megakaryocyte-Induced Fibrosis in Human Bone Marrow Stromal Cells To measure the effects of primed UCMSCs and normal (i.e., non-primed) UCMSCs in the post-ET MF and PMF cell models, in this experiment, human bone marrow stromal cells were co-cultured with SET-2 cells in the presence of IL-1β, followed by treatment with normal UCMSCs or primed UCMSCs and measurement of the expression levels of fibrosis gene markers.

[0098] In summary, human bone marrow stromal cells were divided into four groups, specifically, a normal control group (NCG), a pathological control group (PCG), a comparative group (CG), and an experimental group (EG). The number of human bone marrow stromal cells in each group was 4×10 4cells. Next, human bone marrow stromal cells in each of PCG, CG, and EG were co-cultured with 1×10 5 cells of SET-2 cells pretreated with IL-1β (Note: SET-2 cells were first treated with 30 ng / mL of IL-1β for 24 hours and then IL-1β was removed) for 72 hours, while no treatment was given to human bone marrow stromal cells in NCG. After separating SET-2 cells from human bone marrow stromal cells, human bone marrow stromal cells in CG were treated with normal UCMSCs for 72 hours so that the cell number ratio of normal UCMSCs to human bone marrow stromal cells was 1:2, and human bone marrow stromal cells in EG were treated with priming UCMSCs for 72 hours so that the cell number ratio of priming UCMSCs to human bone marrow stromal cells was 1:2, while no treatment was given to human bone marrow stromal cells in NCG and PCG. Then, after removing normal UCMSCs or priming UCMSCs, total RNA extraction, cDNA synthesis, real-time PCR, normalization of gene expression data, and calculation of the relative mRNA expression levels of COL1A1, FN1, and ACTA2 were performed on human bone marrow stromal cells in each group using the procedures and conditions described in Section A of Example 2 above. The results are shown in Figure 16.

[0099] Figure 16 shows the relative expression levels of (a) COL1A1, (b) FN1, and (c) ACTA2 in human bone marrow stromal cells in each group. As shown in Figure 16, the respective relative expression levels of COL1A1 and FN1 measured in the pathological control group were significantly higher than the levels measured in the normal control group, indicating that fibrosis was induced in human bone marrow stromal cells after co-culture with SET-2 cells pretreated with IL-1β. Furthermore, the respective relative mRNA expression levels of COL1A1 and ATCA2 measured in the comparison group were significantly decreased compared to the levels measured in the pathological control group, indicating that the treatment with normal UCMSCs significantly improved the fibrosis of human bone marrow stromal cells. Furthermore, the relative mRNA expression level of COL1A1 measured in the experimental group was significantly decreased compared to the level measured in the comparison group, indicating that priming UCMSCs enhanced the effect on the fibrosis of human bone marrow stromal cells compared to normal UCMSCs.

[0100] In summary, the above results demonstrated that administration of MSCs to a myelofibrosis mouse model could alleviate anemia, splenomegaly, and myelofibrosis, which are the three major clinical symptoms of myelofibrosis. Furthermore, as shown by the decrease in the relative mRNA expression levels of gene markers of fibrosis and inflammation, MSCs exhibited anti-fibrotic and anti-inflammatory effects in a cellular model of myelofibrosis. Additionally, primed MSCs showed higher efficacy against fibrosis in a cellular model of myelofibrosis compared to normal MSCs (i.e., non-primed MSCs). Therefore, MSCs are expected to be highly likely to be used as a therapeutic agent for alleviating myelofibrosis in myelofibrosis.

[0101] In the above, for the sake of explanation, many specific details have been shown to facilitate a complete understanding of the embodiments. However, it is clear to those skilled in the art that one or more other embodiments can be implemented without showing specific details. Also, in the description indicating "one embodiment" or "an embodiment" in this specification, it should be understood that all descriptions accompanied by designations such as ordinal numbers can be included in specific implementations of the present invention having specific aspects, structures, and features. Furthermore, in this description, sometimes multiple variations are incorporated into one embodiment, drawing, or their descriptions for the purpose of rationalizing the description and for the purpose of understanding the multifaceted nature of the present invention. Also, one or more features or specific examples in one embodiment can, where appropriate, be implemented together with one or more features or specific examples in other embodiments in the implementation of the present disclosure.

[0102] Although the present disclosure has been described in connection with exemplary embodiments, it is understood that the present disclosure is not limited to the disclosed embodiments, but is intended to cover various configurations included within the spirit and scope of the broadest interpretation and to encompass all such modifications and equivalent configurations.

Claims

1. Use of a composition comprising mesenchymal stem cells (MSCs) in the manufacture of a medicament for alleviating myelofibrosis in a subject.

2. The use according to claim 1, wherein the mesenchymal stem cells are selected from the group consisting of umbilical cord-derived mesenchymal stem cells (UCMSCs), bone marrow-derived mesenchymal stem cells (BMSCs), adipose tissue-derived mesenchymal stem cells (AMSCs), dermal-derived mesenchymal stem cells (DMSCs), epidermal-derived mesenchymal stem cells (EMSCs), synovial-derived mesenchymal stem cells (SMMs), dental tissue-derived mesenchymal stem cells (dental MSCs), lung-derived mesenchymal stem cells (LMSCs), and combinations thereof.

3. The use according to claim 2, wherein the mesenchymal stem cells are umbilical cord-derived mesenchymal stem cells.

4. The use according to claim 3, wherein the umbilical cord-derived mesenchymal stem cells are non-primed umbilical cord-derived mesenchymal stem cells.

5. The use according to claim 3, wherein the umbilical cord-derived mesenchymal stem cells are primed umbilical cord-derived mesenchymal stem cells.

6. The use according to claim 5, wherein the primed umbilical cord-derived mesenchymal stem cells are indoleamine pyrrole-2,3-dioxygenase (IDO)-expressing umbilical cord-derived mesenchymal stem cells.

7. The use according to claim 6, wherein the IDO-expressing umbilical cord-derived mesenchymal stem cells are prepared by culturing non-primed umbilical cord-derived mesenchymal stem cells in a culture medium supplemented with IFN-γ.

8. The use according to claim 7, wherein a substance selected from the group consisting of vitamins, steroids, cytokines, double-stranded RNA, and histone deacetylase (HDAC) inhibitors is further added to the medium.

9. The use according to claim 8, wherein the vitamin is retinoic acid, the steroid is dexamethasone or budesonide, the cytokine is TNF-α, the double-stranded RNA is polyinosinic acid-polycytidylic acid, and the histone deacetylase inhibitor is valproic acid.

10. The use according to claim 1, wherein the subject has not received a myeloablative transplant.

11. The use according to claim 1, wherein the myelofibrosis is selected from the group consisting of primary myelofibrosis, myelofibrosis after essential thrombocythemia, myelofibrosis after polycythemia vera, and combinations thereof.

12. The use according to claim 1, wherein a decrease in the level of myelofibrosis or an improvement in inflammation is observed in the subject after administration of the medicament.

13. The use according to claim 12, wherein further improvement in splenomegaly or anemia is observed after administration of the pharmaceutical product to the subject.

14. A method for alleviating myelofibrosis, comprising administering a composition containing mesenchymal stem cells (MSCs) to a subject in need of alleviating myelofibrosis.

15. The method according to claim 14, wherein the mesenchymal stem cells are selected from the group consisting of umbilical cord-derived mesenchymal stem cells (UCMSCs), bone marrow-derived mesenchymal stem cells (BMMMSCs), adipose tissue-derived mesenchymal stem cells (AMSCs), dermal-derived mesenchymal stem cells (DMSCs), epidermal-derived mesenchymal stem cells (EMSCs), synovium-derived mesenchymal stem cells (SMMMSCs), dental tissue-derived mesenchymal stem cells (dental MSCs), lung-derived mesenchymal stem cells (LMSCs), and combinations thereof.

16. The method according to claim 15, wherein the mesenchymal stem cells are umbilical cord-derived mesenchymal stem cells.

17. The method according to claim 16, wherein the umbilical cord-derived mesenchymal stem cells are non-primed umbilical cord-derived mesenchymal stem cells.

18. The method according to claim 16, wherein the umbilical cord-derived mesenchymal stem cells are primed umbilical cord-derived mesenchymal stem cells.

19. The method according to claim 18, wherein the primed umbilical cord-derived mesenchymal stem cells are indoleamine pyrrole-2,3-dioxygenase (IDO)-expressing umbilical cord-derived mesenchymal stem cells.

20. The method according to claim 19, wherein the IDO-expressing umbilical cord-derived mesenchymal stem cells are prepared by culturing non-primed umbilical cord-derived mesenchymal stem cells in a culture medium supplemented with IFN-γ.

21. The method according to claim 20, wherein a substance selected from the group consisting of vitamins, steroids, cytokines, double-stranded RNA, and histone deacetylase (HDAC) inhibitors is further added to the medium.

22. The method according to claim 21, wherein the vitamin is retinoic acid, the steroid is dexamethasone or budesonide, the cytokine is TNF-α, the double-stranded RNA is polyinosinic acid-polycytidylic acid, and the histone deacetylase inhibitor is valproic acid.

23. The method according to claim 14, wherein the subject has not received myeloablative transplantation.

24. The myelofibrosis is the method according to claim 14, selected from the group consisting of primary myelofibrosis, post-essential thrombocythemia myelofibrosis, post-polycythemia vera myelofibrosis, and combinations thereof.

25. The method according to claim 14, wherein a decrease in the level of myelofibrosis or an improvement in inflammation is observed in the subject after administration of the pharmaceutical.

26. The method according to claim 25, wherein further improvement in splenomegaly or improvement in anemia is observed in the subject after administration of the pharmaceutical.

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