Medium composition for inducing erythroid differentiation and use thereof

Abstract

The present invention relates to a medium composition for inducing erythroid differentiation and, more specifically, to: a medium composition for inducing erythroid differentiation; and a method for differentiating or producing erythrocytes, using same. It was confirmed that the medium composition for inducing erythroid differentiation, according to the present invention, can not only be prepared at very low costs as compared to conventional media, but can also rapidly induce the growth, differentiation, and enucleation of erythroid progenitor cells. Therefore, the medium composition for inducing erythroid differentiation, of the present invention, can be effectively used in blood-related clinical fields and therapeutic fields for diseases associated with reduced erythrocytes.

Description

Medium composition for inducing erythroid differentiation and use thereof

[0001] The present invention relates to a medium composition for inducing erythrocyte differentiation, and more particularly, to the medium composition for inducing erythrocyte differentiation; and a method for differentiating or producing erythrocytes using the same.

[0002] Blood transfusions are a crucial treatment for patients with blood shortages for various reasons. However, due to a decline in blood donors and an aging population, donated blood is in short supply. Furthermore, transfusion-related complications, such as the risk of infectious disease transmission, are also a concern. In particular, for red blood cells, a component of blood used for oxygen transport, attempts have been made to use hemoglobin solutions or oxygen carriers as alternatives to the current system of donor blood transfusions. However, these have shown low oxygen-carrying capacity and serious side effects. Consequently, research into ex vivo production of red blood cells is ongoing.

[0003] As a potential alternative to donated blood cells, blood production from various stem cells is being studied. Furthermore, in vitro production of erythrocytes derived from human pluripotent stem (hiPS) cells has been reported, but the resulting erythroblasts suffer from limitations such as inadequate differentiation timing and enucleation. Other studies have also attempted in vitro production of erythrocytes using serum-supplemented media, but these have been limited by the morphology of the resulting erythrocytes, limited proliferation, and low enucleation rates.

[0004] StemSpan TM SFEM II medium promotes the expansion or lineage-specific differentiation of normal or leukemic human hematopoietic stem and progenitor cells (HSPCs), and is widely used as a representative hematopoietic stem cell culture and differentiation medium for mass production of erythroid cells. However, the high manufacturing cost of this medium limits its industrial use in erythroid differentiation processes.

[0005] Therefore, there is a need for new erythroid mass production technologies and culture methods that can promote the growth and erythroid differentiation of erythroid progenitor cells in a more economical manner while replacing the existing expensive differentiation media.

[0006] Accordingly, the inventors of the present invention, while researching a new method for efficiently producing red blood cells, confirmed that a polyvinyl caprolactam-polyvinyl acetate-polyethylene glycol graft copolymer has an effect of inducing red blood cell differentiation, and developed a medium composition for inducing red blood cell differentiation comprising the polyvinyl caprolactam-polyvinyl acetate-polyethylene glycol graft copolymer, and completed the present invention by confirming that the medium composition effectively induces red blood cell differentiation and enucleation.

[0007] Accordingly, an object of the present invention is to provide a composition for inducing erythrocyte differentiation comprising a polyvinyl caprolactam-polyvinyl acetate-polyethylene glycol graft copolymer (PCL-PVAc-PEG).

[0008] Another object of the present invention is to provide an in vitro erythrocyte differentiation or production method, including an erythrocyte differentiation step of culturing erythrocyte progenitor cells using the composition for inducing erythrocyte differentiation.

[0009] Another object of the present invention is to provide red blood cells produced by the above red blood cell production method and an artificial blood composition containing the same.

[0010] To achieve the above purpose, the present invention provides a medium composition for inducing erythrocyte differentiation comprising a polyvinyl caprolactam-polyvinyl acetate-polyethylene glycol graft copolymer (PCL-PVAc-PEG).

[0011] The present invention also provides a medium additive composition for inducing erythrocyte differentiation comprising a polyvinyl caprolactam-polyvinyl acetate-polyethylene glycol graft copolymer.

[0012] In addition, the present invention provides an in vitro erythroid differentiation method including an erythroid differentiation step of culturing erythroid progenitor cells using the medium composition.

[0013] The present invention also provides an in vitro erythrocyte production method comprising an erythrocyte differentiation step of culturing erythrocyte progenitor cells using the medium composition.

[0014] The present invention also provides red blood cells produced by the above red blood cell production method.

[0015] The present invention also provides an artificial blood composition comprising red blood cells produced by the above red blood cell production method.

[0016] The erythroid differentiation-inducing medium composition according to the present invention not only has a significantly lower manufacturing cost than conventional media, but also rapidly induces growth, differentiation, and enucleation of erythroid progenitor cells. Therefore, the erythroid differentiation-inducing medium composition of the present invention can be usefully utilized in blood-related clinical fields and in the treatment of diseases related to erythropoiesis.

[0017] Figure 1 is a diagram showing the results of analyzing the erythrocyte differentiation rate when applying a medium composition for inducing erythrocyte differentiation according to the present invention.

[0018] Figure 2 is a diagram showing the results of confirming the expression of erythrocyte differentiation markers when applying a medium composition for inducing erythrocyte differentiation according to the present invention through flow cytometry.

[0019] Figure 3 is a diagram showing the results of analyzing the erythrocyte denucleation rate when applying a medium composition for inducing erythrocyte differentiation according to the present invention.

[0020] Figure 4 is a diagram showing the results of confirming the expression of erythrocyte differentiation markers and enucleation markers when applying a medium composition for inducing erythrocyte differentiation according to the present invention through flow cytometry analysis.

[0021] Figure 5 is a diagram showing the results of confirming the total number of cells according to the number of days of culture when applying a medium composition for inducing erythrocyte differentiation according to the present invention.

[0022] Figure 6 is a diagram showing the results of calculating the cell growth rate according to the number of days of culture when applying the medium composition for inducing erythrocyte differentiation according to the present invention.

[0023] Figure 7 is a diagram showing the results of microscopic observation of differentiated cells using a medium composition for inducing erythrocyte differentiation according to the present invention.

[0024] Figure 8 is a diagram showing the results of visual observation of a cell pellet obtained by centrifuging differentiated cells using a medium composition for inducing erythrocyte differentiation according to the present invention.

[0025] Hereinafter, the present invention will be described in detail.

[0026] According to an aspect of the present invention, the present invention provides a composition for inducing erythroid differentiation, comprising a polyvinyl caprolactam-polyvinyl acetate-polyethylene glycol graft copolymer (PCL-PVAc-PEG). The composition for inducing erythroid differentiation may be a medium composition or a medium additive composition.

[0027] In a specific example of the present invention, the polyvinyl caprolactam-polyvinyl acetate-polyethylene glycol graft copolymer may be represented by the following chemical formula 1. The polyvinyl caprolactam-polyvinyl acetate-polyethylene glycol graft copolymer is used as a pharmaceutical excipient in the art for the purpose of improving the solubility and bioavailability of poorly soluble drugs during formulation and delivery of drugs.

[0028]

[0029] In a preferred embodiment of the present invention, the PCL-PVAc-PEG is preferably included at a concentration of 0.00001 to 5% (v / v), more preferably 0.0001 to 0.5% (v / v), and even more preferably 0.01 to 0.05% (v / v).

[0030] In the present invention, the medium refers to a liquid covering cells in a culture vessel, such as a petri plate or multi-well plate, and contains nutrients that nourish and support the cells. The culture medium may also include growth factors added to produce desired changes in the cells.

[0031] In a specific example of the present invention, the medium composition is IMDM (Iscove's Modified Dulbecco's Medium) medium, MEM (Minimum Essential Medium), BME (Basal Medium Eagle), DMEM (Dulbecco's Modified EagleMedium), EMEM (Eagle's minimal essential medium), IMDM (Iscove's Modified Dulbecco's Medium), GMEM (Glasgow's MEM), F12 (Ham's F12 Medium), DMEM / F12, RPMI1640, BMOC-3 (Brinster's BMOC-3 Medium), CMRL-1066, L-15 medium (Leibovitz's L-15 medium), McCoy's 5 A, Media 199, MEM α medium, MCDB105, MCDB131, MCDB153, MCDB201, Williams' medium E, Advanced MEM, Advanced DMEM, Advanced It may include at least one basic culture medium selected from the group consisting of DMEM / F-12 and Advanced RPMI1640; and PCL-PVAc-PEG; and preferably, it may include IMDM medium and PCL-PVAc-PEG.

[0032] In a specific embodiment of the present invention, the composition may further include one or more components selected from the group consisting of ITS-X (insulin-transferrin-selenium-ethanolamine), 2-mercaptoethanol, glutamax, ascorbic acid-2-phosphate, and lipid concentrate, and preferably may include all of ITS-X, 2-mercaptoethanol, glutamax, ascorbic acid-2-phosphate, and lipid concentrate.

[0033] The above ITS-X is used as a supplement to the basic medium for the purpose of reducing the amount of fetal bovine serum used in cell culture.

[0034] The above 2-mercaptoethanol is a thiol compound and is widely used to delay the oxidation of biological compounds in solution.

[0035] Glutamax, a dipeptide substitute for L-glutamine, is used in mammalian and stem cell cultures. Glutamax is used to improve growth efficiency in mammalian cell culture systems.

[0036] The above ascorbic acid-2-phosphate is a vitamin derivative that acts as a differentiation inducer in the fields of cell differentiation and tissue engineering.

[0037] The above lipid concentrate is used to remove or replace FBS in cell culture medium and for the purpose of cell growth and maintenance during cell culture.

[0038] In a specific example of the present invention, the induction of erythroid differentiation may be enucleation induction.

[0039] In a specific example of the present invention, the induction of erythroid differentiation may be the induction of differentiation from hematopoietic stem cells or erythroid cells into erythrocytes.

[0040] In the present invention, induction of differentiation refers to a change from a default cell type (genotype and / or phenotype) to a non-default cell type (genotype and / or phenotype). Therefore, "induction of erythroid differentiation" refers to the induction of division of hematopoietic stem cells into progeny cells (i.e., erythrocytes) having characteristics different from those of hematopoietic stem cells, such as genotype (e.g., changes in gene expression determined by genetic analysis) and / or phenotype (e.g., changes in protein expression). In a broad sense, induction of erythroid differentiation of the present invention refers to inducing a change from hematopoietic stem cells or erythroid cells to progeny cells (i.e., erythrocytes) through endothelial-hematopoietic transition, hematopoietic induction, or enucleation induction.

[0041] In the present invention, endothelial-hematopoietic transition refers to the process of transition from stem cells to hematopoietic stem cells.

[0042] The stem cells may be undifferentiated stem cells, i.e., pluripotent stem cells (PSCs) or induced pluripotent stem cells (iPSCs). The pluripotent stem cells may include, without limitation, stem cells isolated or isolated from a patient or donor, or stem cell lines sold commercially. The pluripotent stem cells, also commonly known as PS cells, include any cell that can differentiate into almost any cell, i.e., cells derived from any of the three germ layers (germinal epithelium), including endoderm (inner stomach lining, gastrointestinal tract, lungs), mesoderm (muscle, bone, blood, urogenital), and ectoderm (epithelial tissues and nervous system). PSCs may be derived from embryonic stem cells (including embryonic germ cells) or may be the descendants of totipotent cells obtained by inducing non-pluripotent cells, such as adult somatic cells, by forcing the expression of specific genes.

[0043] In addition, the above-mentioned induced pluripotent stem cells, also commonly abbreviated as iPS cells, refer to a type of pluripotent stem cell that is artificially induced from a normally non-pluripotent cell, such as an adult somatic cell, by inducing the “forced” expression of a specific gene.

[0044] In the present invention, hematopoietic induction means inducing differentiation of hematopoietic stem cells into erythroid progenitor cells.

[0045] In the present invention, enucleation refers to the process by which the nuclei of erythroid progenitor cells gradually condense during differentiation, and the erythroid progenitor cells lose their condensed nuclei. After enucleation, the erythroid progenitor cells become reticulocytes, and through a maturation process, ultimately become erythrocytes.

[0046] The composition for inducing erythrocyte differentiation of the present invention may further include known substances necessary for maintaining, growing, proliferating, and inducing differentiation of cells.

[0047] In a preferred embodiment of the present invention, a medium composition for inducing erythrocyte differentiation can be prepared as described in Example 1 and Table 1 below.

[0048] The erythroid differentiation-inducing medium composition according to the present invention not only has a lower manufacturing cost than conventional media, but also can rapidly induce growth, differentiation, and enucleation of erythroid progenitor cells, as confirmed through examples. Therefore, the erythroid differentiation-inducing medium composition of the present invention can be usefully utilized in blood-related clinical fields and in the field of treatment of diseases related to erythropoiesis.

[0049]

[0050] According to another aspect of the present invention, the present invention provides an in vitro erythrocyte differentiation method, comprising an erythrocyte differentiation step of culturing erythrocyte progenitor cells using the medium composition for inducing erythrocyte differentiation.

[0051] In the present invention, cell culture means in vitro cell growth in an artificial medium for research or medical treatment.

[0052] The culture of the present invention can be carried out using any method known in the art as long as the purpose of the present invention can be achieved, and can be arbitrarily controlled by a person skilled in the art as long as no abnormality is observed in the shape and activity of the cells.

[0053] That is, the culture process of the present invention can be performed according to media and culture conditions known in the art. These culture processes can be easily adjusted and used by those skilled in the art depending on the selected cells.

[0054] The above culture can use a stationary culture or suspension culture method. Stationary culture means culturing in a state where the culture is left alone in the culture medium without agitation or shaking, and suspension culture means culturing in a state where the cells are suspended without being attached to the bottom or side of the reactor through aeration or agitation. In addition, the reactor for stationary culture and the reactor for suspension culture may be the same or different.

[0055] In a specific example of the present invention, the erythrocyte differentiation is preferably performed by differentiating and enucleating erythrocyte progenitor cells into erythrocytes.

[0056] In a specific embodiment of the present invention, the erythroid differentiation step may include (a) culturing erythroid progenitor cells with the erythroid differentiation inducing medium composition further comprising at least one selected from the group consisting of IL-3, SCF (stem cell factor), and EPO (Erythropoietin); (b) culturing erythroid progenitor cells with the erythroid differentiation inducing medium composition further comprising SCF (stem cell factor) or EPO (Erythropoietin); and (c) culturing erythroid progenitor cells with the erythroid differentiation inducing medium composition further comprising EPO (Erythropoietin).

[0057] In a preferred embodiment of the present invention, the culturing in step (a) may be performed for 6 to 12 days, preferably for 7 to 11 days, and more preferably for 8 or 9 days.

[0058] In a preferred embodiment of the present invention, the culturing in step (b) may be performed for 1 to 7 days, preferably for 2 to 6 days, and more preferably for 4 days.

[0059] In a preferred embodiment of the present invention, the culturing in step (c) may be performed for 1 to 5 days, preferably for 2 to 4 days, and more preferably for 3 days.

[0060] In an embodiment of the present invention, erythroid progenitor cells were cultured for 9 days for erythroid differentiation using (a) a culture medium composition for inducing erythroid differentiation further comprising IL-3, SCF, and EPO; (b) erythroid progenitor cells were cultured for 4 days using a culture medium composition for inducing erythroid differentiation further comprising SCF and EPO; and (c) erythroid progenitor cells were cultured using a culture medium composition for inducing erythroid differentiation further comprising EPO.

[0061] The culture methods (a) to (c) of the present invention can be appropriately selected by those skilled in the art as long as they can achieve the purpose of the present invention. That is, the culture period can be determined by confirming that the cultured cells exhibit the desired characteristics.

[0062] In a specific embodiment of the present invention, the erythroid differentiation method may further include a step of 'determining the expression of an erythroid differentiation marker or an enucleation marker.' This is a process for determining whether the cells finally obtained are erythroid cells, and the expression of erythroid differentiation markers (CD71, GPA (CD235a)) and enucleation markers (DRAQ1) can be determined using an analysis method known in the art (e.g., flow cytometry, microscopic observation).

[0063] Typically, red blood cell production occurs through a step-by-step differentiation process derived from hematopoietic stem cells. Hematopoietic stem cells undergo differentiation to become erythroid progenitor cells, undergo nuclear condensation and enucleation, and then mature, ultimately becoming red blood cells. The aforementioned differentiation process typically takes place in vitro for 21 days. The present invention's method for inducing red blood cell differentiation accelerates the differentiation process, with enucleation rapidly increasing by day 13 of culture. Therefore, the method for inducing red blood cell differentiation of the present invention can enhance the efficiency of red blood cell production.

[0064]

[0065] According to another aspect of the present invention, there is provided a method for producing erythrocytes in vitro, comprising an erythrocyte differentiation step of culturing erythrocyte progenitor cells using the erythrocyte differentiation-inducing medium composition. The present invention also provides erythrocytes produced by the method, and an artificial blood composition comprising the same.

[0066] The erythrocyte production method of the present invention may further include a step of "determining the expression of erythrocyte differentiation markers or enucleation markers." This step is a process for determining whether the cells ultimately obtained are erythrocytes. The expression of erythrocyte differentiation markers (CD71, GPA (CD235a)) and enucleation markers (DRAQ1) is confirmed using an analytical method known in the art (e.g., flow cytometry).

[0067] Typically, erythroid differentiation occurs in vitro for 21 days. During this differentiation process, cell proliferation is limited after erythroid progenitor cells, specifically polychromatic erythroblasts, mature erythrocytes. Therefore, mass cell proliferation is a crucial factor in erythrocyte production. Through examples, it was confirmed that the number of erythrocytes produced using the erythrocyte production method of the present invention was more than twice that of cells produced using conventional media.

[0068] The red blood cells produced using the red blood cell production method of the present invention can be utilized in the form of artificial blood compositions, etc. The artificial blood composition of the present invention can be transfused into a subject and can contribute to resolving issues such as blood shortages and transfusion side effects. Furthermore, the produced red blood cells and artificial blood composition can be usefully utilized in blood-related clinical fields and in the treatment of diseases related to red blood cell reduction.

[0069]

[0070] Duplicate contents are omitted in consideration of the complexity of this specification, and terms not otherwise defined in this specification have meanings commonly used in the technical field to which the present invention belongs.

[0071] Hereinafter, the present invention will be described in more detail through examples. These examples are intended solely to illustrate the present invention, and it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples.

[0072] [Experimental Example]

[0073] Experimental Example 1. hiPSC Culture

[0074] CMC-hiPSC-011 cells, obtained from the National Stem Cell Bank, were maintained in Essential 8 (E8) medium (Gibco A1517001) in iMatrix-511 (Matrixome, 892 021) and fed daily. hiPSCs were dissociated into clumps using ReLeSR (Stemcell Tech., 05873), passaged, and reseeded in E8 medium containing Y-27632 (2 μM; Stemcell Tech, 72307), a p160-ROCK (Rho-associated coiled-coil kinase) inhibitor.

[0075]

[0076] Experimental Example 2. Endothelial-to-hematopoietic transition (EHT) stage after mesoderm induction

[0077] Induced pluripotent stem cells (iPSCs) were maintained in Essential 8 (E8) medium (Gibco A1517001) in iMatrix-511 (Matrixome, 892 021). Cells were detached using TrypLE (Gibco 12563011) and cultured in mTeSR™3D medium (StemCell Technologies 3950) according to the manufacturer's instructions on an orbital shaker set at 70 rpm for 2 days. Pluripotent spheroids were then transferred to STEMdiff™ APEL™2 medium containing 10 μM CHIR-99021 (Selleckchem), 50 ng / ml BMP4 (Miltenyi), and 50 ng / ml VEGF-165 (Miltenyi) and cultured for 2 days. After removing CHIR99021, the cells were cultured for 2 days. Finally, the cells were cultured for 2 days in the presence of 50 ng / ml BMP4 (Miltenyi), 50 ng / ml VEGF-165 (Miltenyi), and 50 ng / ml SCF (Miltenyi). After 6 days of mesoderm induction, 20 embryoid bodies (EBs) were transferred to 35 mm gelatin-coated dishes (Sigma G1393) and cultured with medium for 4 days. The medium contains STEMdiff APEL2 Medium (STEMCELL Technologies) 5% PFHM-II Protein-Free Hybridoma Medium (Thermo Fisher Scientific), 10 ng / ml BMP4, 100 ng / ml SCF, 100 ng / ml FLT3-L (Miltenyi), 50 ng / ml TPO (Miltenyi), 50 ng / ml VEGF-165, 25 ng / ml IL-3 (PeproTech), 25 ng / ml IL-6 (PeproTech), 10 ng / ml IGF-II (PeproTech) and 10 ng / ml FGF-2 (R&D Systems).Afterwards, the medium was replaced with STEMdiff APEL2 medium (STEMCELL Technologies), 5% PFHM-II Protein-Free Hybridoma Medium (Thermo Fisher Scientific), 100 ng / ml SCF, 100 ng / ml FLT3-L, 50 ng / ml TPO, 25 ng / ml IL-3, and 25 ng / ml IL-6.

[0078]

[0079] Experimental Example 3. Hematopoietic induction

[0080] After 8 days of EHT, cells were cultured in hematopoietic induction medium for 7 days until erythroid progenitors were induced. The hematopoietic induction medium contained STEMdiff APEL2 Medium (STEMCELL Technologies), 5% PFHM-II Protein-Free Hybridoma Medium (Thermo Fisher Scientific), 5 ng / ml IL-3, 100 ng / ml SCF, 3 U / ml EPO (PeproTech), and 1 μM Dexamethasone (Stem Cell Technologies). For erythroid differentiation, the hematopoietic-induced cell suspension (i.e., erythroid progenitor cells) was harvested.

[0081]

[0082] Experimental Example 4. Erythroid differentiation

[0083] The erythroid progenitor cells (i.e., hematopoietic-induced cells) harvested in the above Experimental Example 3 were divided into an experimental group, a positive control group, and a negative control group, and an erythroid differentiation test was performed. Specifically, the erythroid progenitor cell suspension (7.5 x 10) harvested in the above Experimental Example 3 4cells / ml) were cultured for 13 days according to the three-phase erythropoiesis protocol. Cells were cultured in Soluplus-enhanced IMDM media from day 0 to day 13. StemSpan™ SFEM II media and Splus base without Soluplus were used as positive and negative controls, respectively.

[0084] In addition to the aforementioned medium, differentiation was induced using medium containing 5 ng / ml IL-3, 100 ng / ml SCF (stem cell factor), and 3 U / ml EPO (erythropoietin) from day 0 to day 8. In addition, the culture was continued by replacing the medium with 100 ng / ml SCF and 3 U / ml EPO from day 8 to day 11. Finally, differentiation was induced by replacing the medium with 3 U / ml EPO from day 11 to day 13. The culture period can be extended to 18 days if necessary. Hematopoietic and erythroid differentiation was confirmed using cell counting, flow cytometry, and microscopic evaluation at designated intervals.

[0085] Recombinant human EPO, IL-3, and SCF used in this experiment were purchased from PEPROTECH.

[0086]

[0087] Experimental Example 5. Cell Counting

[0088] The number of viable cells was counted using trypan blue staining. Hematopoietic and erythrocytes were harvested, centrifuged, and the supernatant was removed. The cells were then suspended in medium and washed. The suspended cells were stained 1:1 with trypan blue solution and counted using a cell counter (LunaII cell counter, Logos Biosystems).

[0089]

[0090] Experimental Example 6. Flow Cytometry

[0091] To analyze the expression of surface protein markers of in vitro differentiated erythrocytes, they were labeled with the following anti-human antibodies: CD71-PE (BD Pharmingen), glycophorin A (GPA)-FITC (BD Pharmingen), and immunoglobulin G2 IgG2-FITC and IgG2-PE (BD Pharmingen) were used for isotypic control staining.

[0092] Cells were washed with PBS, resuspended in PBS / 5% fetal bovine serum, and reacted with 10 μl of each monoclonal antibody at 4°C for 30 min. Cells were washed twice with PBS / 5% FBS, resuspended in 300 μl formaldehyde (2%), and analyzed using a BD FACSAria II (BD Bioscience). Flow cytometry data were analyzed using FlowJo™ Software (BD Bioscience).

[0093]

[0094] Experimental Example 7. Microscopic Evaluation

[0095] The erythroid differentiation pattern of hematopoietic stem cells was observed using an inverted microscope (Ts2-FL, Nikon).

[0096]

[0097] [Example]

[0098] Example 1. Preparation of a medium composition for inducing erythroid differentiation

[0099] Soluplus enhanced IMDM medium was prepared for erythroid differentiation. The above Soluplus enhanced IMDM medium contained Iscove's liquid medium (GIBCO, Cat No. 12440053), 0.01% or 0.05% Soluplus (BASF), 1X Insulin-Transferrin-Selenium-Ethanolamine (ITS-X) (GIBCO, Cat No. 51500056, 100X ITS-X was purchased and diluted with 1X ITS-X for use), Glutamax (GIBCO, Cat No. 35050061), 50 μg / ml l-ascorbic acid 2-phosphate (Vc2P) (Sigma, Cat No. A8960), 1:1000 β-mercaptoethanol (GIBCO, Cat No. 21985023), and 0.05% Chemically Defined Lipid Concentrate (GIBCO, Cat No. Includes 11905031).

[0100] In addition, as a positive control, StemSpan™ SFEM II medium (Stem cell technologies, Cat No. 9655), which is widely used in the field for hematopoietic stem cell culture and differentiation, was used as a base medium. The StemSpan™ SFEM II medium is supplemented with 10% AB serum (Innovative Research, ISERAB100ML) and 330 mg / mL transferrin (R&D Systems, Cat No. 2914-HT-001G). The negative control (Splus base) is a medium that does not contain Soluplus, and other components are identical to the Soluplus-enhanced IMDM medium.

[0101] The medium composition for inducing erythrocyte differentiation described above was prepared as shown in Table 1 below, and the negative control group was prepared as shown in Table 2.

[0102] Soluplus Enhanced IMDM MediumIscove's MDM98 mlSoluplus(10% stock)0.001%(v / v) or 0.05%(v / v)1 Defined Lipid Concentrate(starting from day 8)1:500

[0103] Splus base medium (negative control)Iscove's MDM98 mlITSX (insulin-transferrin-selenium-ethanolamine)1 ml2-Mercaptoethanol100 ulGlutamax1 mlAscorbic acid 2 phosphate (50ug / ml)100ulCD lipid concentrate(starting from day 8)1:500

[0104] Example 2. Confirmation of the effect of inducing erythroid differentiation according to the concentration of Soluplus in the medium composition for inducing erythroid differentiation.

[0105] In order to elucidate the potential effect of Soluplus on the maturation and enucleation of erythroid progenitor cells, this example confirmed the effect of inducing erythroid differentiation according to the presence or absence of Soluplus and its concentration.

[0106]

[0107] 2-1. Differentiation rate

[0108] CD71 and GPA (glycophorin A) are used as surface markers to detect the differentiation of erythroid cells into erythroid cells. Therefore, the erythroid differentiation rate according to the presence or absence of Soluplus and its concentration was investigated through CD71+ / GPA+ expression analysis. Specifically, cells were cultured in Soluplus-enhanced IMDM medium (soluplus 0.01% or 0.05%). The negative control (splus base) is a medium with 0% soluplus concentration. Cells were harvested on days 7 and 13 of culture and subjected to flow cytometry to confirm the expression of erythroid differentiation markers (CD71, GPA (CD235a)). The expression ratio of CD71 and GPA (i.e., differentiation rate) was calculated based on the marker expression analysis results, and the results of flow cytometry are shown in Figure 1, and the results of flow cytometry are shown in Figure 2.

[0109] As shown in Figures 1 and 2, the Soluplus-enhanced IMDM medium group showed a significantly higher differentiation rate of erythroid progenitor cells than the negative control group (Splus base), and a higher differentiation effect was confirmed as the concentration of Soluplus increased. In particular, the Soluplus-enhanced IMDM medium 0.05% group showed the highest differentiation rate at 63.9% on day 7.

[0110]

[0111] 2-2. Enucleation rate

[0112] The enucleation rate according to the presence or absence of Soluplus and its concentration was investigated through expression analysis of the erythroid differentiation marker (GPA (CD235a)) and the enucleation marker (DRAQ1). Specifically, cells were cultured in Soluplus-enhanced IMDM medium (soluplus 0.01% or 0.05%). The negative control (splus base) is a medium with a 0% concentration of soluplus. Cells were harvested on days 7 and 13 of culture and subjected to flow cytometry to confirm the expression of the erythroid differentiation marker (GPA (CD235a)) and the enucleation marker (DRAQ1). The expression ratio of DRAQ1 and GPA (i.e., enucleation rate) was calculated based on the results of marker expression analysis, and the results of flow cytometry are shown in Figure 3, and the results of flow cytometry are shown in Figure 4.

[0113] As shown in Figures 3 and 4, the Soluplus-enhanced IMDM medium group had a significantly higher enucleation rate than the negative control group (Splus base). In particular, the Soluplus-enhanced IMDM medium 0.01% group had the highest enucleation rate at 35.79% on the 13th day.

[0114] Through the above results, it was confirmed that Soluplus has the effect of inducing differentiation and enucleation of erythroid progenitor cells into erythroid cells.

[0115]

[0116] Additionally, the effects of inducing erythroid differentiation using Soluplus enhanced IMDM medium (0.05%) or a positive control (StemSpan™ SFEM II medium) were compared. Specifically, the differentiation and enucleation rates were compared and analyzed through analysis of the expression of erythroid differentiation markers (CD71, GPA (CD235a)) and enucleation markers (DRAQ1).

[0117] As a result, the Soluplus-enhanced IMDM medium group showed differentiation rates 2.8-fold and 5.8-fold higher on days 7 and 13, respectively, than the positive control group using StemSpan™ SFEM II medium, a conventional erythroid differentiation medium. In addition, the Soluplus-enhanced IMDM medium group was confirmed to have enucleation rates 274-fold and 6.2-fold higher on days 7 and 13, respectively, than the positive control group.

[0118] The above results indicate that when the Soluplus-enhanced IMDM medium is applied to erythroid differentiation, the differentiation rate and enucleation rate are significantly higher than those of the medium not containing Soluplus and the medium conventionally used for erythroid differentiation.

[0119]

[0120] Example 3. Confirmation of cell proliferation effect of medium composition for inducing erythroid differentiation

[0121] 3-1. Total cell number and cell proliferation

[0122] Induction of erythrocyte differentiation and mass proliferation are very important factors in erythrocyte production. Therefore, the cell proliferation effect according to the use of Soluplus enhanced IMDM medium (0.05%) was confirmed. The positive control (StemSpan™ SFEM II medium) was used as the control group in this experiment. To confirm the cell proliferation effect, the total cell number was confirmed by counting cells according to the method of Experimental Example 5, and the cell growth rate was calculated based on the cell count results. The total cell number and cell growth rate are shown in Figures 5 and 6, respectively.

[0123] As shown in Figures 5 and 6, the total cell number and cell growth rate in the Soluplus-enhanced IMDM medium (0.05%) group and the positive control group were similar at the beginning of culture (day 4). However, in the later stages of culture, the total cell number and cell growth rate in the Soluplus-enhanced IMDM medium (0.05%) group were confirmed to be higher than those in the positive control group. In particular, the cell growth rate in the Soluplus-enhanced IMDM medium (0.05%) group was twice as high as that in the positive control group on the 10th day of culture. In other words, it was confirmed that the use of the Soluplus-enhanced medium can effectively induce not only erythroid differentiation but also cell proliferation.

[0124]

[0125] 3-2. Observation of cells differentiated into red blood cells

[0126] Cells differentiated into erythrocytes were observed under a microscope using the method of Experimental Example 7. Furthermore, cells differentiated into erythrocytes were harvested to obtain a cell suspension, which was then centrifuged to obtain a cell pellet. The resulting cell pellet was observed visually. The results of microscopic observation of differentiated cells are shown in Figure 7, and the results of visual observation of the cell pellet are shown in Figure 8.

[0127] As shown in Figure 7, smooth, round cells were observed in the Soluplus-enriched IMDM medium (0.05%) group (arrows). These smooth, round cells are fully mature erythrocytes that have undergone the enucleation process. In contrast, no such cells were observed in the positive control group.

[0128] As shown in Fig. 8, the cell pellet of the Soluplus-enhanced IMDM medium (0.05%) group was confirmed to be larger than that of the positive control group. This means that the number of red blood cells ultimately obtained in the Soluplus-enhanced IMDM medium (0.05%) group was greater.

[0129]

[0130] In summary, the present inventors have developed a medium composition for inducing erythroid differentiation (i.e., Soluplus enhanced IMDM medium). The medium composition for inducing erythroid differentiation according to the present invention is not only cheaper to manufacture than conventional media, but also rapidly induces growth, differentiation, and enucleation of erythroid progenitor cells. Therefore, the medium composition for inducing erythroid differentiation of the present invention can be usefully utilized in blood-related clinical fields and in the treatment of diseases related to erythropoiesis.

[0131]

[0132] While specific aspects of the present invention have been described in detail, it will be apparent to those skilled in the art that these specific descriptions merely represent preferred embodiments and are not intended to limit the scope of the present invention. Therefore, the substantial scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A medium composition for inducing erythroid differentiation comprising polyvinyl caprolactam-polyvinyl acetate-polyethylene glycol graft copolymer (PCL-PVAc-PEG).

2. A composition according to claim 1, wherein the PCL-PVAc-PEG is included at a concentration of 0.00001 to 5% (v / v).

3. In the first paragraph, the medium composition is IMDM (Iscove's Modified Dulbecco's Medium) medium, MEM (Minimum Essential Medium), BME (Basal Medium Eagle), DMEM (Dulbecco's Modified EagleMedium), EMEM (Eagle's minimal essential medium), IMDM (Iscove's Modified Dulbecco's Medium), GMEM (Glasgow's MEM), F12 (Ham's F12 Medium), DMEM / F12, RPMI1640, BMOC-3 (Brinster's BMOC-3 Medium), CMRL-1066, L-15 medium (Leibovitz's L-15 medium), McCoy's 5 A, Media 199, MEM α medium, MCDB105, MCDB131, MCDB153, MCDB201, Williams' medium E, Advanced MEM, Advanced DMEM, Advanced At least one basic culture medium selected from the group consisting of DMEM / F-12 and Advanced RPMI1640; and A composition comprising PCL-PVAc-PEG; 4. A composition according to claim 1, wherein the composition further comprises at least one component selected from the group consisting of ITS-X (insulin-transferrin-selenium-ethanolamine), 2-mercaptoethanol, glutamax, ascorbic acid-2-phosphate, and lipid concentrate.

5. A composition according to claim 1, wherein the induction of erythrocyte differentiation is enucleation induction.

6. A composition according to claim 1, wherein the induction of erythroid differentiation is induction of differentiation from hematopoietic stem cells or erythroid cells into erythrocytes.

7. A composition of a medium additive for inducing erythrocyte differentiation comprising a polyvinyl caprolactam-polyvinyl acetate-polyethylene glycol graft copolymer.

8. A method for inducing erythroid differentiation in vitro, comprising an erythroid differentiation step of culturing erythroid progenitor cells using the medium composition of paragraph 1.

9. A method according to claim 8, wherein the erythrocyte differentiation is performed by differentiating and enucleating erythrocyte progenitor cells into erythrocytes.

10. In the 8th paragraph, the erythrocyte differentiation step is (a) a step of culturing erythroid progenitor cells using the medium composition of claim 1, further comprising at least one selected from the group consisting of IL-3, SCF (stem cell factor), and EPO (Erythropoietin); (b) a step of culturing erythroid progenitor cells with the medium composition of the first paragraph further comprising SCF (stem cell factor) or EPO (Erythropoietin); and (c) A method comprising a step of culturing erythroid progenitor cells with the medium composition of the first clause further comprising EPO (Erythropoietin).

11. A method according to claim 10, wherein the culturing in step (a) is performed for 6 to 12 days.

12. A method according to claim 10, wherein the culturing in step (b) is performed for 1 to 7 days.

13. A method according to claim 10, wherein the culturing in step (c) is performed for 1 to 5 days.

14. An in vitro erythrocyte production method comprising an erythrocyte differentiation step of culturing erythrocyte progenitor cells using the medium composition of paragraph 1.

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