Composition for inducing differentiation of erythroid precursor cells

Abstract

The present invention relates to a composition for inducing differentiation of erythroid precursor cells, and more specifically, to: a composition for inducing differentiation of erythroid precursor cells comprising extracellular vesicles derived from macrophages; and a method for inducing differentiation into erythrocytes by treating erythroid precursor cells with macrophage-derived extracellular vesicles.

Description

Technical Field

[0001] This invention relates to a composition for inducing differentiation of erythroid precursor cells, comprising extracellular vesicles derived from macrophages.

[0002] This application claims priority to Korean Patent Application No. 10-2023-0165958, filed with the Korean Intellectual Property Office on November 24, 2023, the entire disclosure of which is incorporated herein by reference. Background Technology

[0003] Blood transfusion is a vital treatment for emergencies and chronic blood disorders such as anemia, myelodysplastic syndromes, and leukemia. However, due to the hyperimmunization problem caused by repeated transfusions and the anticipated shortage of qualified blood donors in the context of a global aging population, the transfusion issue is expected to become a significant public health concern in the future. Currently, in vitro erythropoiesis research is being conducted based on stem cell types, but further investigation into the normal mechanisms of erythropoiesis is needed because the efficiencies of proliferation, differentiation, and enucleation differ from those in vivo.

[0004] Meanwhile, all cells exchange information with other cells or the external environment. To facilitate this information transmission, cells secrete various substances into the external environment, among which representative soluble factors include cytokines, chemokines, hormones, and neurotransmitters. In recent years, in addition to soluble factors, information exchange through extracellular vesicles (EVs) has gradually attracted attention. EVs are nanoscale vesicles encased in a lipid bilayer, secreted into the external environment by all cells.

[0005] Extracellular vesicles (EVs) contain proteins, lipids, various types of RNA and DNA, and can be relatively easily isolated from various biological fluids such as blood, cerebrospinal fluid (CSF), urine, amniotic fluid, breast milk, saliva, and semen. Specific proteins are highly concentrated in EVs, making them easy to detect. These EVs contain a variety of different components depending on the cell type from which they originate, and analysis of vesicle components can serve as biomarkers for early disease diagnosis or as indicators of patient treatment response. Furthermore, EVs themselves can serve as carriers for therapeutic agents. Due to these characteristics and applicability, research on EVs has accelerated in recent years.

[0006] In particular, macrophage-derived EVs are known to play a crucial role as mediators in the tumor microenvironment (TME) and have the ability to promote intercellular and organ-to-cell communication. This is mainly attributed to their ability to carry various types of bioactive molecules (including nucleic acids and proteins) that are essential for communication processes.

[0007] Therefore, despite recent attention to EV research, there is still a lack of studies on the effects of EV on erythropoiesis. Furthermore, while the interaction between macrophages and erythroid precursor cells in the erythroblastic islands of the bone marrow is known to be crucial, the underlying mechanisms have not been fully elucidated.

[0008] Existing research aimed at in vitro erythrocyte differentiation has limitations in terms of yield. Therefore, the inventors conducted in-depth research to improve erythrocyte differentiation efficiency and discovered that macrophage-derived EVs promote erythroid precursor cell differentiation and regulate the erythrocyte formation process, thus leading to this invention. Summary of the Invention

[0009] Technical issues

[0010] The present invention aims to provide a composition for inducing differentiation of erythroid precursor cells, comprising macrophage-derived EVs.

[0011] The present invention also aims to provide a method for inducing differentiation of erythroid precursor cells, which includes culturing erythroid precursor cells in a culture medium containing macrophage-derived EVs.

[0012] Technical solution

[0013] The inventors have dedicated themselves to the in vitro production of erythrocytes and the improvement of erythrocyte differentiation yield in vitro, in order to solve the problem of blood supply shortage required for surgical procedures or other purposes. As a result, when erythroid precursor cells were treated with macrophage-derived EVs, it was found that the EVs could promote the differentiation of erythroid precursor cells into erythrocytes and regulate erythropoiesis.

[0014] Therefore, the present invention provides a composition comprising macrophage-derived EVs for inducing erythroid precursor cell differentiation. The composition of the present invention for inducing differentiation can be used to induce erythroid precursor cell differentiation and maturation into erythrocytes, and to regulate the erythropoiesis process.

[0015] In this invention, "progenitor cells" refer to undifferentiated cells that possess self-renewal and differentiation capabilities but ultimately differentiate into predetermined cell types. Progenitor cells exhibit a predetermined differentiation pathway but typically do not express markers of mature, fully differentiated cells, or possess the functions of mature, fully differentiated cells. Therefore, progenitor cells differentiate into relevant cell types, but under normal conditions, they usually cannot form a diverse range of cell types. This invention utilizes erythroid precursor cells.

[0016] In this invention, "erythroid precursor cells" refers to all cells involved in erythropoiesis (excluding fully mature erythrocytes), such as hematopoietic stem cells or unenucleated erythroid cells. For example, erythroid precursor cells can be CD34 cells. +Erythroid precursor cells can be selected from the following groups: burst-forming unit-erythroid (BFU-E), colony-forming unit-erythroid (CFU-E), proerythroblasts, basophilic erythroblasts, polychromatic erythroblasts, orthochromatic erythroblasts, and reticulocytes. Erythroid precursor cells can be obtained from various sources, such as peripheral blood, cord blood, or bone marrow.

[0017] In this invention, "extracellular vesicles (EVs)" refer to nanoscale membrane structures produced and secreted by various cell types according to the physiological or pathological state of the cell. These vesicles contain a variety of biomolecules produced by the cell and play an important role in intercellular communication by transmitting information. Specifically, these vesicles are surrounded by a lipid bilayer derived from the cell membrane and may contain proteins, lipids, RNA, DNA, and other metabolites. These EVs can be classified into exosomes, extracellular bodies / microvesicles, etc., based on their biosynthetic mechanisms.

[0018] In this invention, macrophages, as members of the immune system, act as part of a non-specific defense mechanism, regulate inflammatory responses, and secrete important cytokines and chemokines that promote tissue repair and regeneration. These secreted substances activate other neighboring immune cells or participate in intercellular signaling, thereby regulating the intensity and duration of the immune response. Furthermore, macrophages possess antigen-presenting capabilities, displaying pathogen-derived antigens on their cell surface and activating specific immune cells such as T cells.

[0019] When tissues are damaged or infected, macrophages can transform into two main activation states, commonly referred to as M1 and M2. M1 macrophages are pro-inflammatory macrophages that promote antibacterial and tumor-killing responses, while M2 macrophages are involved in promoting anti-inflammatory responses and tissue repair.

[0020] In this invention, "macrophage-derived EV" can refer to EV secreted by macrophages and includes bioactive substances related to the functional characteristics of macrophages.

[0021] In one embodiment of the invention, macrophage-derived EVs may originate from M2 macrophages. Therefore, the present invention discloses the effect of EVs, particularly those secreted by M2 macrophages, on erythropoiesis.

[0022] In this invention, "differentiation" refers to the process by which cells, structures, or tissues lacking a specific function or morphology divide and grow to acquire a mature structure or specific function. For example, in this invention, it refers to the process by which immature cells develop into mature red blood cells. Furthermore, in this invention, "erythropoiesis" refers to the process by which hematopoietic stem cells develop into mature red blood cells, and during this erythropoiesis process, the growth, differentiation, and maturation of erythroid precursor cells occur.

[0023] In this invention, macrophage-derived EVs can induce erythroid precursor cells to differentiate and mature into erythrocytes, and regulate erythropoiesis. Furthermore, in this invention, macrophage-derived EVs can regulate various factors in the erythropoiesis and differentiation process, such as cell cycle, differentiation rate, hormones, iron availability, cell membrane composition and structure, or the intracellular and extracellular environment.

[0024] In this invention, macrophage-derived EVs can induce erythroid precursor cell differentiation and regulate erythropoiesis through direct cell-cell interaction via contact with erythroid precursor cells. Macrophage-derived EVs can also induce erythroid precursor cell differentiation through direct internalization into erythroid precursor cells or by activating intracellular signaling pathways via cell adhesion molecules, but this invention is not limited to these methods. Furthermore, macrophage-derived EVs can induce erythroid precursor cell differentiation through non-contact methods via intracellular signal transduction or gene expression regulation, but this invention is not limited to these methods either.

[0025] The results of observing EVs secreted by macrophages after fluorescent labeling confirmed that EVs were directly internalized into erythroid precursor cells.

[0026] Furthermore, in this invention, it has been demonstrated that macrophage-derived EVs increase the expression of cell adhesion molecules. In this invention, "cell adhesion molecules (CAMs)" or "adhesion proteins," as proteins present on the cell surface, refer to molecules that mediate adhesion between cells or between cells and the extracellular matrix, and play a key role in biological processes such as cell-cell interactions, cell migration, inflammatory responses, wound healing, tissue formation, immune responses, and metastasis.

[0027] Specifically, in this invention, by verifying the expression of erythroid hematopoietic island-associated adhesion proteins such as macrophage erythrocyte adherent protein (MAEA), intercellular adhesion molecule 4 (ICAM4), erythropoietin receptor (EPOR), and α-hemoglobin stabilizing protein (AHSP), it was confirmed that the expression level of adhesion proteins in erythroid precursor cells treated with macrophage-derived EVs was significantly increased compared to the control.

[0028] In this invention, MAEA, or erythrocyte macrophage protein (EMP), is a protein that mediates the interaction between erythroid precursor cells and macrophages. This protein promotes the adhesion of erythroid precursor cells to central macrophages within erythroid hematopoietic islands, thus playing a crucial role in erythrocyte maturation and differentiation. ICAM4 is an adhesion molecule present on the surface of erythrocytes, mediating adhesion between erythrocytes and endothelial cells and regulating erythrocyte migration within blood vessels and into tissues. EPOR is a receptor located on the surface of erythroid precursor cells that binds to erythropoietin (EPO) (a hormone that regulates erythropoiesis) and activates signaling pathways that promote the survival, maturation, and differentiation of erythroid precursor cells. AHSP binds to α-globulin, thereby reducing the toxicity of globulin chains that cannot independently transport oxygen and protecting them before they assemble into hemoglobin. Therefore, this protein plays a crucial role in the efficient production and maturation of erythrocytes. The aforementioned adhesion proteins MAEA, ICAM4, EPOR, or AHSP correspond to adhesion proteins expressed during the differentiation phase of erythroid precursor cells. Upregulation of these adhesion proteins can be used to determine whether erythroid precursor cell differentiation has been induced. For example, these adhesion proteins can be expressed during the differentiation of proerythrocytes into proerythroblasts.

[0029] In one embodiment of the invention, treatment of macrophage-derived EVs during erythroid precursor cell proliferation was used to confirm whether adhesion protein expression increased. The results showed that the expression levels of ICAM4 and AHSP in the EV-treated group were significantly higher than those in the control group. Therefore, the composition for inducing differentiation containing macrophage-derived EVs of the present invention can be effectively used to induce erythroid precursor cell differentiation.

[0030] Furthermore, in one embodiment of the present invention, when erythroid precursor cells are treated with macrophage-derived EVs, the enucleation rate of the erythroid precursor cells increases, and the expression of the protein markers GPA and CD71 expressed in mature erythrocytes increases. Therefore, the composition for inducing differentiation containing macrophage-derived EVs of the present invention can be effectively used for the differentiation and maturation of erythroid precursor cells.

[0031] The macrophage-derived EVs contained in the compositions of the present invention may have a diameter of 50 nm to 1000 nm, for example 50 nm to 800 nm, 50 nm to 700 nm, 50 nm to 600 nm, 50 nm to 500 nm, 50 nm to 400 nm, 50 nm to 300 nm, 50 nm to 250 nm, 100 nm to 250 nm, 100 nm to 230 nm, 150 nm to 230 nm, or 200 nm to 230 nm.

[0032] Furthermore, in this invention, 1×10 can be used in the composition. 1Up to 1×10 4 EVs / cell, 5×10 1 Up to 1×10 4 EV / cell or 5×10 1 Up to 2×10 3 The concentration of EVs per cell includes EVs derived from macrophages.

[0033] In this invention, erythroid precursor cells can be differentiated into erythrocytes by treating them with a composition containing macrophage-derived EVs for inducing differentiation and culturing them in a differentiation and maturation medium. The culture medium used in this invention can be a minimum cell culture medium (CCMM) containing only carbon, nitrogen, and trace elements, used as a medium for obtaining erythrocytes from erythroid precursor cells in vitro. That is, it is a basal medium, a mixture containing essential sugars, amino acids, and water required for cell survival, but without serum, nutrients, or various growth factors. For example, the minimum cell culture medium can be Duchenne modified Eagle's medium (DMEM), endothelial cell differentiation medium (EDM), minimum essential medium (MEM), Eagle's basal medium (BME), RPMI 1640, F-10, F-12, α-minimum essential medium (α-MEM), Glasgow minimum essential medium (G-MEM), or Iscove modified Duchenne medium.

[0034] In addition, the culture medium used in this invention may contain vitamin C, which can maintain stability against oxidative stress in vitro, and may further contain antibiotics, such as penicillin, streptomycin or gentamicin, if necessary.

[0035] The culture medium used in this invention may further contain at least one component selected from the group consisting of: stem cell factor (SCF), IL-1, IL-3, IL-4, IL-5, IL-11, granulocyte-macrophage colony-stimulating factor (GM-CSF), macrophage colony-stimulating factor (M-CSF), granulocyte colony-stimulating factor (G-CSF), and erythropoietin (EPO) to promote the differentiation and maturation of erythroid precursor cells. More preferably, the culture medium used in this invention contains hydrocortisone, SCF, IL-3, and EPO during the differentiation stage from proerythroblasts to early erythroblasts; contains SCF, IL-3, and EPO during the differentiation stage from early erythroblasts to intermediate erythroblasts; and contains EPO during the differentiation stage from intermediate erythroblasts to late erythroblasts. However, the culture medium does not contain cytokines during the differentiation stage from late erythroblasts to intermediate erythroblasts (i.e., reticulocytes) (i.e., the enucleation stage).

[0036] The culture environment can be any suitable environment for in vitro induction of erythroid precursor cell differentiation. For example, it can be cultured at 5% CO2 and 36 to 38°C (preferably 37°C).

[0037] In one embodiment of the invention, erythroid precursor cells treated with macrophage-derived EVs can be co-cultured with stromal cells. The stromal cells can be mesenchymal stem cells (MSCs), MS-5 cells, or OP-9 cells, but the invention is not limited thereto. In another embodiment of the invention, erythroid precursor cells treated with macrophage-derived EVs can differentiate independently in the culture medium without co-culturing with stromal cells. For example, when erythroid precursor cells differentiate independently in the culture medium, no additional isolation step from co-cultured cells is required, thus allowing independent in vitro induction of erythrocyte differentiation without a separate cell separation or filtration process.

[0038] In addition, the present invention provides a method for inducing differentiation of erythroid precursor cells, comprising culturing erythroid precursor cells in a culture medium containing macrophage-derived EVs.

[0039] All content relating to the composition used for inducing differentiation can be directly applied, with the necessary modifications, to the method of inducing erythroid precursor cell differentiation.

[0040] This invention provides a method for differentiating and maturing erythrocytes by inducing differentiation of erythroid precursor cells.

[0041] Beneficial effects

[0042] This invention provides a method for inducing erythroid precursor cells to differentiate into erythrocytes by treating them with a composition for inducing erythroid precursor cell differentiation (including macrophage-derived EVs) or macrophage-derived EVs. Therefore, this invention, by providing a novel method for the in vitro production or differentiation of erythrocytes, can meet the clinical needs for transfusion blood. Attached Figure Description

[0043] Figure 1 schematically illustrates the culture medium protocols used in macrophage polarization and erythroid precursor cell proliferation and differentiation. Figure 1a M2 polarization of macrophages induced from human monocyte THP-1 cells to M0; Figure 1b Umbilical cord erythrocyte proliferation protocol using EVs. Figure 1c (Umbilical cord erythrocyte differentiation protocol using EVs).

[0044] Figure 2a Histogram showing the particle size distribution of EVs in the supernatant of isolated THP-1-derived M2 macrophages.

[0045] Figure 2bImages of EV distribution in PBS are shown (screenshots from video recorded using Malvern NanoSight LM10 (top) and NanoSight NTA 3.4 (bottom)).

[0046] Figure 2c This is a graph illustrating the average size of EVs and their mode distribution in THP-1 derived macrophages.

[0047] Figure 3 shows the results obtained by measuring the expression of M2 markers (VEGFA, CD169, CD163 and STAT6) and specific adhesion factors (VCAM1 and EMP) in erythroid hematopoietic islands using quantitative real-time PCR.

[0048] Figure 4a A set of CLSM images of red blood cells (green) and EVs (red) 24 hours after EV treatment.

[0049] Figure 4b The image shows the migration of red blood cells (green) and EVs (red) using live-cell imaging 1 hour after EV treatment.

[0050] Figure 5 shows the growth rate, aggregation, and survival rate of erythroid precursor cells after treatment with different concentrations of EV according to the present invention.

[0051] Figure 6 shows the expression patterns of erythroid island-associated adhesion proteins EMP, ICAM4, EPOR, and AHSP at 24 and 72 hours after treatment with different concentrations of EV according to the present invention.

[0052] Figure 7 shows the expression of GPA and CD71 by flow cytometry after treating erythroid precursor cells with different concentrations of EV according to the present invention.

[0053] Figure 8 shows the expression of GPA and CD49d by flow cytometry after treating erythroid precursor cells with different concentrations of EV according to the present invention.

[0054] Figure 9a Histograms showing the conversion of GPA and CD71 expression to mean fluorescence intensity (MFI) after treating erythroid precursor cells with different concentrations of EV according to the present invention are displayed.

[0055] Figure 9b and 9c The diagram shows a comparison of cell morphology and maturation status on day 2 and day 4 after treatment of erythroid precursor cells with different concentrations of EV according to the present invention, following Wright-Giemsa staining. Figure 9bThe pink arrow indicates early erythroblasts, and the red arrow indicates reticulocytes. Detailed Implementation

[0056] The present invention will be described in detail below with reference to specific embodiments. The following embodiments are for illustrative purposes only, and the content of the present invention is not limited to the following embodiments.

[0057] Example

[0058] 1. Preparation of EVs derived from M2 macrophages

[0059] 1) M2 macrophage culture and EV collection

[0060] To continuously produce macrophage-derived EVs, human monocyte THP-1 cells were induced into M0 macrophages and then further polarized into M2 macrophages. First, 4 × 10⁻⁶ cells were... 5 THP-1 cells / mL were continuously differentiated into M0 macrophages in RPMI-1640 medium (Gibco, Roswell Park Memorial Institute: RPMI1640) supplemented with heat-inactivated 10% fetal bovine serum (FBS), 1% penicillin-streptomycin, and 185 ng / mL phorbol 12-myristate 13-acetate (PMA) for 24 hours. Subsequently, M0 macrophages were treated with RPMI 1640 medium supplemented with 50 ng / mL recombinant human M-CSF and 20 ng / mL recombinant human IL-4 for 48 hours, thereby polarizing into M2 ( Figure 1a ).

[0061] After inducing human mononuclear THP-1 cells to M2, they were cultured in RPMI-1640 medium for 24 hours. The supernatant was collected, and EVs were then isolated. To remove cell debris from the supernatant, the cell culture medium was centrifuged at 3000 g for 15 minutes, and the supernatant was purified using ExoQuic according to the manufacturer's instructions. TM -TC (System Biosciences) was treated at 4°C for at least 12 hours, then isolated, diluted with PBS, and stored at -80°C.

[0062] 2) Analysis of EVs derived from M2 macrophages

[0063] The size, distribution, and concentration of isolated THP-1 M2 macrophage-derived EVs were confirmed by nanoparticle tracking analysis (NTA) (Figure 2).

[0064] First, after introducing a portion of EVs into the NTA, the Brownian motion of the particles on each x, y, and z axis was measured, confirming that exosomes were generally distributed between 150 nm and 250 nm. After NTA analysis of some EVs from various samples, the mean size was 208 nm, and the mode was 137.62 nm (Student's t-test). p < 0.0001). NTA size distribution analysis showed that the mode mean was between 100 nm and 150 nm, indicating that exosomes were the main form of EVs, while other forms such as ectosomes or microvesicles also existed.

[0065] 3) Validation of adhesion protein expression in EVs derived from M2 macrophages

[0066] M2 polarization was analyzed by RT-qPCR analysis of the mRNA expression of M2-specific macrophage markers (CD163, CD169, STAT6, VEGFA) and adhesion molecules (VCAM1, MAEA) that form macrophage-erythroid hematopoietic islands. Primers used for the macrophage and erythrocyte markers in the analysis are shown in Table 1 below.

[0067] Table 1

[0068]

[0069] By confirming the changes in the expression of M2 markers (VEGFA, CD169, CD163 and STAT6), it was confirmed that VEGFA was significantly increased by 2.54 times, and both CD169 and STAT6 were expressed (n=4-5, Student's t test). p<0.05. The M2 surface marker CD163 was not detected in M0, but was improved in M2. This confirms that macrophages have appropriately differentiated into the M2 type. Figure 3a Furthermore, as specific adhesion-related factors for erythroid precursor cells and macrophages in erythroid hematopoietic islands, adhesion molecules VCAM1 and EMP (MAEA) were detected at the mRNA level, and they were maintained as adhesion molecules for the formation of erythroid hematopoietic islands even after polarization (n=5-7, Student's t-test). p<0.05. This confirms that the EV donor cells, macrophages, have appropriately differentiated into M2 ( Figure 3b ).

[0070] 2. Verify whether EVs derived from M2 macrophages are internalized by erythroid precursor cells.

[0071] EVs were fluorescently labeled, and z-axis stacking imaging was performed using confocal laser scanning microscopy (CLSM) 24 hours later to confirm whether they were internalized by erythroid precursor cells.

[0072] After the isolated EVs were stained with exosome-specific dyes, to verify whether the EVs were internalized, they were stained with 2×10⁻⁶ dyes under standard proliferation medium conditions. 2 and 1×10 3 24 hours after EV / cell concentration treatment, the results were analyzed using CLSM and compared with a control group not treated with EV. Red-labeled EVs were internalized in a concentration-dependent manner by GFP-expressing erythroid precursor cells, and the resulting cells were confirmed by Z-axis stacking imaging analysis to have EVs localized in the cytoplasm and at elevated concentrations around aggregated erythroid precursor cells (Figure 4). These results indicate that EVs at 2 × 10⁻⁶ cells / cell concentrations are effective at increasing concentrations. 2 Up to 1×10 3 It is internalized by erythroid precursor cells within the (EV / cell) concentration range.

[0073] Furthermore, within 3 hours post-EV injection, enhanced marker fluorescence expression due to erythroid precursor cell migration and EV internalization was observed using CLSM live-cell imaging. Specifically, 1 × 10⁻⁶ markers were used 1 hour prior to measurement. 3 Following EV / cell concentration injection, CLSM-based live-cell imaging was employed, acquiring Z-axis stacked images every 5 minutes for 3 hours to observe migrating and interacting erythroid precursor cells in a quiescent state over time. Here, red-labeled EVs are localized in the cytoplasm, with their intensity increasing over time. During the 3-hour observation period, approximately 1 hour later, erythroid lineage cells exhibited an aggregation pattern, with their number increasing 1.5-fold compared to the initial field of view.

[0074] 3. Co-culture of erythroid precursor cells and EVs derived from macrophages

[0075] Erythroid precursor cells were analyzed under proliferation and differentiation culture conditions. Under proliferation culture conditions, cells were cultured in StemSpan supplemented with 1% penicillin-streptomycin (Gibco), 3 IU / mL EPO (Calbiochem, San Diego, CA), 50 ng / mL SCF (R&D Systems, Minneapolis, MN), 1 μM DEX, and 1 μg / mL doxycycline (Takara, Shiga, Japan). tm Cultured in STEMCELL Technologies medium. A control without EV treatment and cultures treated with 1×50 and 1×10⁻⁶ EVs respectively. 2 and 2×10 2 The experimental groups treated with EV / cell concentrations were cultured for 30 days, with the differentiation medium changed every 2-3 days. Similarly, under the same proliferation medium conditions, EV treatment (0, 2 × 10⁻⁶ cells / cells) was performed. 2 1×10 3 and 2×10 3EVs / cells), then incubated for 24 hours and 72 hours.

[0076] For the basal differentiation medium conditions, Iscove modified Duchenne broth (IMDM; Gibco, Waltham, MA) was prepared as the basal medium and supplemented with 10% tetracycline-free fetal bovine serum, 10 μg / mL insulin, 200 μg / mL transferrin, and 1% penicillin-streptomycin (Gibco), and then filtered through a 20-μm filter. From day 0 to 2, the basal medium was supplemented with cytokines (3 U / mL EPO, 10 ng / mL SCF, and 1 ng / mL IL-3); and from day 2 to 4, 3 U / mL EPO and 10 ng / mL SCF were added. The medium protocols for proliferation and differentiation are shown in Figure 1.

[0077] 4. Validation of erythrocyte proliferation induced by EVs derived from M2 macrophages

[0078] 1) Verification of proliferation trend

[0079] Immortalized human erythroid precursor cell lines were cultured in proliferation medium for 30 days, while simultaneously being treated with different concentrations of EV (0, 1×50, 1×10⁻⁶). 2 and 2×10 2 The cells were treated with EVs (e.g., EVs / cells). The results showed that higher EV concentrations resulted in lower cumulative cell numbers. Figure 5a Furthermore, aggregation of erythroid precursor cells was observed in the later stages of culture. Figure 5b This phenomenon was not observed during the initial subculture.

[0080] 2) Toxicity assessment

[0081] In addition, to evaluate the toxicity of macrophage-derived EVs, immortalized human erythroid precursor cell lines were cultured in proliferation medium for 30 days and treated with different concentrations of EVs (0, 1×50, 1×10⁻⁶). 2 and 2×10 2 EVs (cells) are processed.

[0082] The results confirmed that cell viability remained consistently maintained regardless of changes in EV concentration. Figure 5c ).

[0083] 3) Verify whether EVs derived from M2 macrophages increase the expression of cell adhesion molecules.

[0084] To verify whether macrophage-derived EVs induce upregulation of cell adhesion molecule expression, different concentrations of EVs (0, 2 × 10⁻⁶ ...) were injected. 2 1×10 3 and 2×10 3After EV / cell assay, the expression patterns of erythroid hematopoietic island-associated adhesion proteins EMP, ICAM4, EPOR, and AHSP in erythroid precursor cells were detected by RT-qPCR at 24 and 72 hours.

[0085] The results confirmed that during a 24-hour treatment, 2×10 3 At EV / cell concentration, the expression of cell adhesion molecule ICAM4 increased 2.5-fold compared to the control. Figure 6a ); and in the 72-hour processing, 2×10 3 At EV / cell concentrations, the expression of AHSP, a gene specifically expressed in erythroid lineage cells, was increased 6-fold compared to the control. Furthermore, at 2×10⁻⁶ cells / cell concentrations... 3 At EV / cell concentration (instead of 2×10) 2 EV / cell), AHSP expression was significantly increased by 3.15-fold ( Figure 6b ).

[0086] 5. Validation of EV-induced erythroid precursor cell differentiation from M2 macrophages

[0087] 1) Verify the expression of proteins (GPA and CD71) in erythroid precursor cells using EVs derived from M2 macrophages.

[0088] EV was administered at different concentrations (0, 2×10⁻⁶). 2 1×10 3 Erythroid precursor cells were treated with erythroid precursor cell differentiation medium to differentiate into erythrocytes. The enucleation rate of EV-treated erythroid precursor cells and the levels of proteins (GPA and CD71) expressed in mature erythrocytes were assessed by flow cytometry and Giemsa staining. Results on day 2 and day 4 of differentiation were validated.

[0089] First, by verifying the expression of GPA and CD71 on day 2 of differentiation between the control and EV-treated experimental groups, the results showed no difference in the expression of the erythroid markers GPA and CD71. Figure 7a On day 4 of differentiation, the average expression of GPA and CD71 markers in the EV-treated experimental group increased by 10% (); Figure 7b ), and after converting to a mean fluorescence intensity (MFI) histogram, the experimental group also observed these increases ( Figure 9a That is, it was confirmed that EVs derived from M2 macrophages contribute to the expression of specific markers in preenucleated mature erythrocytes.

[0090] On the other hand, no difference was observed between the control and experimental groups on day 2 of differentiation for CD49d, whose expression decreases as it differentiates into mature erythrocytes. Figure 8a However, on day 4 of differentiation, the proportion of GPA(+) / CD49d(+) was higher in the EV-treated experimental group. Figure 8bThe results indicate that the overall erythrocyte-specific GPA expression was relatively high and strong in the experimental group, while the proportion of immature erythrocytes was also high.

[0091] 2) Verification of enucleation of erythroid precursor cells using EVs derived from M2 macrophages

[0092] Furthermore, by comparing the enucleation rates of the control and EV-treated experimental groups, the results confirmed that there was no significant change in the enucleation rate between the control and EV-treated experimental groups on day 2 of differentiation; however, on day 4 of differentiation, the proportion of erythroblasts to late erythroblasts in the experimental group was higher than that in the control.

[0093] Specifically, morphology and maturity were compared after Giemsa staining on days 2 and 4 of differentiation. Figure 9b There were no differences between the experimental groups on day 2 of differentiation, but on day 4, the proportion of proerythroblasts in the EV-treated experimental groups was similar to that in the control (control: 31.73%; 2×10⁻⁶). 2 EV / cell: 28.33%; 1×10 3 EV / cell ratio: 30.70%. Meanwhile, regarding the proportion of intermediate erythroblasts, the control group showed 41.01%, which was higher than the experimental group (2×10⁻⁶). 2 EV / cell: 30.34%, 1×10 3 EV / cell ratio: 35.04%. However, regarding the proportion of late erythroblasts, the control group showed 24.58%, which was lower than the EV-treated experimental group (2×10⁻⁶). 2 EV / cell: 39.04%, 1×10 3 EV / cell: 30.45% Figure 9c ).

[0094] The present invention has been described with reference to the above embodiments and experimental examples, but this is only an exemplary description. Those skilled in the art should understand that various modifications and equivalent embodiments and experimental examples can be made accordingly. Therefore, the actual scope of protection of the present invention should be determined by the technical spirit of the appended claims.

Claims

1. A composition for inducing differentiation of erythroid precursor cells, comprising extracellular vesicles derived from macrophages.

2. The composition according to claim 1, wherein the extracellular vesicles derived from macrophages promote the differentiation of erythroid precursor cells into erythrocytes.

3. The composition according to claim 1, wherein the macrophage is an M2 macrophage.

4. The composition according to claim 1, wherein the extracellular vesicles derived from macrophages are directly internalized into the erythroid precursor cells.

5. The composition used according to claim 1, wherein the composition increases the expression of ICAM4 and / or AHSP in the erythroid precursor cells.

6. The composition used according to claim 1, wherein the composition increases the expression of GPA and / or CD71 in the erythroid precursor cells.

7. The composition according to claim 1, wherein the extracellular vesicles derived from macrophages have a diameter ranging from 50 nm to 1000 nm.

8. The composition according to claim 1, wherein the composition contains a concentration of 1×10 1 EV / cell up to 1×10 4 Extracellular vesicles derived from macrophages in EV / cells.

9. The composition used according to claim 1, wherein the composition further comprises EPO, SCF or IL-3.

10. The composition according to claim 1, wherein the erythroid precursor cells are selected from the group consisting of hematopoietic stem cells, bursting erythroid colony-forming units (BFU-E), erythroid colony-forming units (CFU-E), proerythrocytes, early erythroblasts, intermediate erythroblasts, late erythroblasts, and reticulocytes.

11. A method for inducing differentiation of erythroid precursor cells, comprising culturing erythroid precursor cells in a culture medium containing extracellular vesicles derived from macrophages.

12. The method of claim 11, further comprising additional treatment of EPO, SCF, or IL-3.

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