Errα gene-deficient erythrocyte progenitor cells and method for differentiating erythrocytes thereof

Abstract

The present invention relates to ERRα gene-deficient erythrocyte progenitor cells and a method for differentiating erythrocytes thereof. It was confirmed that ERRα gene-deficient cells, in which ERRα genes are knocked out in erythrocyte progenitor cells, exhibit a higher expression level of erythrocyte differentiation markers and a more rapid progression of erythrocyte differentiation under erythrocyte differentiation conditions according to the present invention. Accordingly, the present invention provides a method for rapidly differentiating erythrocytes by using ERRα gene-deficient erythrocyte progenitor cells.

Description

ERRα gene-deficient erythrocyte progenitor cells and methods for differentiating them into erythrocytes

[0001] The present invention relates to erythrocyte progenitor cells with a deficiency in the ERRα gene and a method for differentiating the same into erythrocytes. The present invention was carried out under Project No. KH140743 with the support of the Ministry of Health and Welfare of the Republic of Korea, the managing agency for the said project is the Korea Health Industry Development Institute, and the project title is "Development of Novel Erythrocyte Differentiation and Proliferation Technology through Elucidation of Differentiation Dynamics of High-Efficiency Mobilized Hematopoietic Stem Progenitor Cells Based on Nisch Enhancement Factor." Additionally, the present invention was carried out under Project No. 2022R1A5A2027161 with the support of the Ministry of Science and ICT of the Republic of Korea, the managing agency for the said project is the National Research Foundation of Korea, and the project title is "Molecular Control Research Center for Cancer Cell Diversity." In addition, the present invention was carried out under project number 00333287 with the support of the Ministry of Science and ICT of the Republic of Korea, the managing agency of the said project is the National Research Foundation of Korea, and the project title is "Research on the control of differentiation induction of immature leukemia / cancer stem cells based on novel differentiation-promoting factors in the bone marrow microenvironment and research on the treatment of blood cancer."

[0002] Red blood cells are the cells that make up the majority of the blood cells, accounting for 44% of all blood cells, and primarily perform the function of transporting oxygen. Red blood cells are produced from hematopoietic stem cells through several differentiation stages, passing through the burst forming unit erythroid (BFU-E), colony forming unit erythroid (CFU-E), proerythroblast, basophilic erythroblast, polychromatic erythroblast, and orthochromatic erythroblast. As the nucleus gradually condenses, the cell size decreases. After the condensed nucleus enucleates, it becomes a reticulocyte; when the remaining RNA and microorganelles disappear and it takes on a biconcave shape, it becomes a mature erythrocyte (RBC). This differentiation process generally takes 3 weeks in vitro, and cell proliferation is limited after the polychromatic erythroblast stage, which is a mature erythroblast.

[0003] Red blood cells are essential in emergency situations such as hemorrhage, as well as in pathological medical conditions involving cancer, blood disorders, and surgery. While red blood cells are collected globally through blood transfusions, there are limitations to the availability of cells under suitable conditions. Furthermore, as issues such as contamination by pathogens and viruses arise during the collection and transportation of blood, various attempts are being developed to provide safe blood. One such approach is the production of artificial blood, the basis of which is the in vitro production of red blood cells. A known method for producing red blood cells in vitro utilizes a differentiation medium for regulatory stem cells. However, current methods face critical limitations for large-scale production and suffer from the problem of inadequate red blood cell maturation.

[0004] The object of the present invention is to produce ERRα gene-deficient erythrocyte progenitor cells and provide a method for differentiating them into erythrocytes.

[0005] The present invention provides a method for differentiating red blood cells, comprising the steps of: knocking out the ERRα (estrogen-related receptor alpha) gene in red blood cell progenitor cells in vitro; and culturing the red blood cell progenitor cells with the knocked-out ERRα gene in a red blood cell differentiation medium to differentiate the red blood cell progenitor cells into red blood cells.

[0006] In addition, differentiated red blood cells according to the above method are provided.

[0007] According to the present invention, it has been confirmed that ERRα gene-deficient cells, in which the ERRα gene is knocked out in erythrocyte progenitor cells, express erythrocyte differentiation markers at a higher rate and undergo faster erythrocyte differentiation under the erythrocyte differentiation conditions according to the present invention, thereby providing a method to rapidly differentiate erythrocytes using ERRα gene-deficient erythrocyte progenitor cells.

[0008] Figure 1 is an experimental schematic for establishing an ERRα gene-deficient cell line in erythrocyte progenitor cells using the CRISPR-Cas9 system.

[0009] Figure 2 is the result of analyzing whether the ERRα gene is deleted in ERRα gene-deficient cells established according to the present invention using Quantitative RT-PCR.

[0010] FIG. 3 is an experimental schematic for differentiating ERRα gene-deficient erythrocyte progenitor cells established according to the present invention into erythrocytes.

[0011] Figure 4 is the result of analyzing the change in the expression level of erythrocyte differentiation markers after differentiating ERRα gene-deficient erythrocyte progenitor cells established according to the present invention into erythrocytes.

[0012] The terms used in this specification have been selected based on currently widely used general terms whenever possible, taking into account their functions in the present invention; however, these terms may vary depending on the intent of those skilled in the art, case law, the emergence of new technologies, etc. Additionally, in specific cases, terms have been arbitrarily selected by the applicant, and in such cases, their meanings will be described in detail in the relevant description of the invention. Therefore, the terms used in this invention should be defined not merely by their names, but based on their meanings and the overall content of the invention.

[0013] Unless otherwise defined, all terms used herein, including technical or scientific terms, have the same meaning as generally understood by those skilled in the art to which the present invention pertains. Terms such as those defined in commonly used dictionaries should be interpreted as having a meaning consistent with their meaning in the context of the relevant technology, and should not be interpreted in an ideal or overly formal sense unless explicitly defined in this application.

[0014] The present invention will be described in more detail below.

[0015] The present invention provides a method for differentiating red blood cells, comprising the steps of: knocking out the ERRα (estrogen-related receptor alpha) gene in red blood cell progenitor cells in vitro; and culturing the red blood cell progenitor cells with the knocked-out ERRα gene in a red blood cell differentiation medium to differentiate the red blood cell progenitor cells into red blood cells.

[0016] The aforementioned progenitor cell is an undifferentiated cell with self-replicating and differentiation capabilities, but it is an ultimately differentiated cell in which the type of cell to which it ultimately differentiates is already determined.

[0017] The above erythroid precursor cell refers to all cells involved in the process of erythropoiesis, excluding mature erythrocytes. Specifically, the above erythroid precursor cell may refer to an erythroid cell prior to enucleation, and may be an immature erythroid precursor cell or a pre-enucleation cell positive for Glycophorin A, a molecule specific to the erythrocyte lineage. For example, the above erythroid precursor cell may be selected from the group consisting of proerythroblasts, basophilic erythroblasts, polychromatic erythroblasts, and orthochromatic erythroblasts.

[0018] The above erythrocyte progenitor cells are cultured in a medium containing SCF (stem cell factor), EPO (erythropoietin), DOX (doxycycline), and DEX (dexamethasone).

[0019] The step of deleting the ERRα gene involves knocking out the ERRα gene (Genebank ID: NC_000011.10) denoted by SEQ ID No. 1 using a CRISPR (Clustered regularly interspaced short palindromic repeats) / Cas9 system with shRNA, and the ERRα shRNA is introduced into erythrocyte progenitor cells via a lentivirus.

[0020] Red blood cell progenitor cells with the above-mentioned ERRα gene are selected in a medium containing puromycin.

[0021] In the step of deleting the ERRα gene, a lentivirus containing sgRNA that binds complementarily to the ERRα gene represented by SEQ ID NO. 1 is added to the erythrocyte progenitor cells and cultured for 24 to 48 hours.

[0022] The step of differentiating into red blood cells comprises: culturing red blood cell progenitor cells with the ERRα gene deficient in EPO (erythropoietin), insulin (insuline), transferrin, DOX (doxycycline), and SCF (stem cell factor) for 4 days in a first differentiation medium containing EPO (erythropoietin), insulin (insuline), transferrin, and DOX (doxycycline) for 3 days in a second differentiation medium containing EPO (erythropoietin), insulin (insuline), transferrin, and DOX (doxycycline); and culturing red blood cell progenitor cells with the ERRα gene deficient in EPO (erythropoietin), insulin (insuline), and transferrin in a second differentiation medium containing EPO (erythropoietin), insulin (insuline), and transferrin.

[0023] The step of differentiating the above red blood cell progenitor cells into red blood cells is completed within 7 to 22 days.

[0024] In addition, differentiated red blood cells according to the above method are provided.

[0025] Hereinafter, experimental examples and embodiments will be described in detail to aid in understanding the present invention. However, the following experimental examples and embodiments are merely illustrative of the content of the present invention and the scope of the present invention is not limited to the following experimental examples and embodiments. The experimental examples and embodiments of the present invention are provided to more completely explain the present invention to those with average knowledge in the art.

[0026]

[0027] [Example 1] Establishment of ERRα gene-deficient cells

[0028] To confirm the role of the ERRα (urea transporter 2) gene in erythrocyte differentiation, an ERRα gene-deficient cell line was established in the human erythrocyte progenitor cell line HUDEP-2 using the CRISPR-Cas9 system in the manner shown in Fig. 1. HUDEP-2 cells were fed Stemspan SFEM (Stemspan containing 0.4 μg / ml dexamethasone (DEX) (Sigma), 1 μg / ml doxycycline (DOX) (Clontech), 50 ng / ml SCF (stem cell factor) (R&D systems), and 5 ng / ml EPO (erythropoietin) (Peprotech). TMThey were cultured in Serum-Free Expansion Medium (STEM CELL technologies). To knock out the ERRα gene in HUDEP-2 cells grown in cytokine-containing medium, HUDEP-2 cells were transformed by spin infection using an ERRα shRNA lentivirus. After adding 0.8 μg / ml of polybrene (Santacruz), a chemical that aids in viral infection, to cytokine-containing Stemspan SFEM medium, 1 x 10⁶ were added to 2 mL of the medium. 6 HUDEP-2 cells were suspended. After dispensing into a 6-well plate, 2 x 10⁶ cells containing ERRα shRNA (sc-44706-SH, Santa Cruz) 6 1 mL of lentivirus at TU / ml was added. Centrifugation was performed for 90 minutes at 20°C and 2,000 rpm. After 24 hours, the cells were resuspended in 2 mL of fresh cell culture medium containing polybrene, 1 mL of ERRα shRNA lentivirus was added, and centrifugation was performed once more. After 24 hours, the medium was replaced with Stemspan SFEM medium containing cytokines. After culturing for 24 hours, the medium was replaced with one containing 0.5 μg / ml puromycin, and the cells were cultured for 5 days to select only virus-infected cells.

[0029] To confirm whether the selected HUDEP-2 cells were genetically modified with ERRα compared to a control group without viral phenotype, the presence of ERRα gene deletion was evaluated via quantitative RT-PCR. RNA was extracted from the cells using the RNeasy plus mini kit (Qiagen). The extracted RNA was synthesized into cDNA using the SmartGene Compact cDNA Synthesis Kit (SMART GENE). The primers used were the ERRα forward primer (SEQ No. 2, CCA CTA TGG TGT GGC ATC CTG T), the ERRα reverse primer (SEQ No. 3, GGT GAT CTC ACA CTC GTT GGA G), the GAPDH forward primer (SEQ No. 4, TGT TGC CAT CAA TGA CCC CTT), and the GAPDH reverse primer (SEQ No. 5, CTC CAC GAC GTA CTC AGC). As shown in Figure 2, quantitative RT-PCR confirmed that the expression of the ERRα gene was reduced in HUDEP-2 cells treated with the CRISPR-Cas9 system compared to control cells that were not transfected with the virus.

[0030]

[0031] [Example 2] Differentiation of erythrocytes in ERRα gene-deficient cells

[0032] The differentiation ability of the above-mentioned ERRα gene knockout erythrocyte progenitor cells into erythrocytes was evaluated. To differentiate the erythrocyte progenitor cells into erythrocytes, the erythrocyte progenitor cells were cultured for 4 days in IMDM (Iscove's Modified Dulbecco's Medium) containing 5% human AB serum (Sigma), 400 μg / mL holo-transferrin (Sigma), 10 μg / mL human insulin (Sigma), 5 ng / mL EPO (erythropoietin) (Peprotech), 1 μg / mL DOX (doxycycline) (Clontech), 41.25 μg / mL heparin (Sigma), and 50 ng / mL SCF (stem cell factor) (R&D systems). Subsequently, the cells were cultured for 3 days after removing SCF from the IMDM medium, and thereafter in a medium without DOC. Erythrocyte differentiation ability was assessed from the onset of differentiation (Day 0) to Days 4, 8, and 12. After treatment with fluorescent antibodies, differences in the expression of the erythrocyte differentiation markers CD235a and CD71 were analyzed via flow cytometry. As shown in Figure 4, compared to the control group, the erythrocyte differentiation marker CD235a in ERRα gene-deficient cells (ERRα shRNA) + and CD71 + It was confirmed that the expression of was at a higher rate and that red blood cell differentiation proceeded more rapidly.

[0033] Foregoing, specific parts of the present invention have been described in detail. It is evident to those skilled in the art that such specific descriptions are merely preferred embodiments and do not limit the scope of the invention. That is, the actual scope of the invention is defined by the appended claims and their equivalents.

[0034] Numerical ranges include the values ​​defined in the above ranges. All maximum numerical limits given throughout this specification include all lower numerical limits as clearly written. All minimum numerical limits given throughout this specification include all higher numerical limits as clearly written. All numerical limits given throughout this specification will include all better numerical ranges within a wider numerical range, as clearly written.

Claims

1. A step of knocking out the ERRα (estrogen-related receptor alpha) gene in erythrocyte progenitor cells in vitro; and A method for differentiating red blood cells comprising the step of culturing red blood cell progenitor cells having a deficiency of the ERRα gene in a red blood cell differentiation medium to differentiate the red blood cell progenitor cells into red blood cells.

2. A method for differentiating red blood cells according to claim 1, characterized in that the red blood cell progenitor cells are cultured in a medium containing SCF (stem cell factor), EPO (erythropoietin), DOX (doxycycline), and DEX (dexamethasone).

3. A method for differentiating red blood cells according to claim 1, wherein the step of deleting the ERRα gene is characterized by knocking out the ERRα gene represented by SEQ ID No. 1 (Genebank ID: NC_000011.10) using a CRISPR (Clustered regularly interspaced short palindromic repeats) / Cas9 system using shRNA.

4. A method for erythrocyte differentiation according to claim 3, characterized in that the ERRα shRNA is introduced into erythrocyte progenitor cells via a lentivirus.

5. A method for differentiating red blood cells according to claim 1, characterized in that the red blood cell precursor cells having a deleted ERRα gene are selected in a medium containing puromycin.

6. In claim 1, the step of deleting the ERRα gene is, A method for differentiating red blood cells, characterized by adding a lentivirus containing sgRNA that binds complementarily to the ERRα gene represented by SEQ ID NO. 1 to the above red blood cell progenitor cells and culturing for 24 to 48 hours.

7. In paragraph 1, the step of differentiating into red blood cells is, A step of culturing erythrocyte progenitor cells deficient in the above ERRα gene for 4 days in a primary differentiation medium containing EPO (erythropoietin), insulin (insuline), transferrin, DOX (doxycycline), and SCF (stem cell factor); A step of culturing erythrocyte progenitor cells deficient in the ERRα gene for 3 days in a secondary differentiation medium containing EPO (erythropoietin), insulin (insuline), transferrin, and DOX (doxycycline); and A method for differentiating red blood cells, characterized by comprising the step of culturing red blood cell progenitor cells having the above-mentioned ERRα gene in a secondary differentiation medium containing EPO (erythropoietin), insulin, and transferrin.

8. A method for differentiating red blood cells according to claim 1, characterized in that the step of differentiating the red blood cell progenitor cells into red blood cells is completed within 7 to 22 days.

9. Red blood cells differentiated by the method of any one of paragraphs 1 to 8.

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