A method for promoting terminal differentiation of red blood cells and its use

Abstract

The application provides a method for promoting terminal differentiation of red blood cells and application thereof. The method is achieved by adding an inhibitor (+)‑JQ1 or MS436 of BRD4 in the process of inducing terminal differentiation of red blood cells. The method for promoting terminal differentiation of red blood cells is reasonable in design and simple in operation. The method is suitable for the differentiation process of red blood cells of different cell sources and can improve the maturity of in-vitro induced red blood cells. The method can promote the differentiation of a red blood cell leukemia cell line TF‑1 and provides a treatment strategy for red blood cell differentiation abnormality caused red blood cell related diseases.

Description

Technical Field

[0001] This invention relates to the field of cell engineering, and in particular to a method for promoting terminal differentiation of erythrocytes and its application. Background Technology

[0002] Red blood cells are the most numerous cells in the body, responsible for transporting oxygen. Abnormal red blood cell differentiation can lead to a variety of erythroid-related diseases, such as different types of anemia, polycythemia vera, M6 erythroid leukemia, and myelodysplastic syndrome. Inducing these abnormally differentiated red blood cells to continue differentiating is an effective way to treat these diseases. Approximately one-third of the global population suffers from red blood cell diseases to varying degrees, seriously affecting patients' health and quality of life. Besides red blood cell diseases, in vitro red blood cell generation is also a major scientific challenge in regenerative medicine, and its solution could help alleviate the increasingly serious blood shortage crisis.

[0003] In vitro production of red blood cells not only provides a new source of blood supply but also helps ensure blood safety, protecting it from the influence of infectious diseases and other factors; it also facilitates the timely fulfillment of transfusion needs for patients with rare blood types. Significant breakthroughs have been achieved in the clinical application of CAR-T cells, CAR-NK cells, and CAR-Mac cells, induced from stem cells, which have been used clinically to treat leukemia, lymphoma, and other malignant hematological diseases with good results. This has brought further hope for the in vitro production of red blood cells from stem cells. However, current in vitro red blood cell production faces challenges such as low red blood cell maturity and difficulties in large-scale production.

[0004] Erythrocyte formation involves a series of complex cell fate-changing events. Hematopoietic stem cells (HSPCs) undergo early and late erythroid progenitor cell colony-forming units to generate primitive erythrocytes. These primitive erythrocytes then undergo several mitotic divisions to successively form early, intermediate, and late erythroblasts. Late erythroblasts enucleate to form reticulocytes and ultimately mature erythrocytes. The process of primitive erythrocytes generating late erythroblasts is crucial for erythrocyte maturation and enucleation, and is known as terminal erythroid differentiation.

[0005] Bromodomain-containing protein 4 (BRD4) is an important member of the BET family and a crucial epigenetic and transcriptional regulatory protein, playing a vital role in normal cell growth and cell cycle progression. BRD4 also plays a significant role in early embryonic development, stem cell maintenance, cancer development, and immunotherapy. Several inhibitors have been developed targeting BRD4, and their single-agent or combined use with other tumor inhibitors has demonstrated good anti-tumor effects. Among them, (+)-JQ1 (molecular formula: C23H25ClN4O2S; CAS: 1268524-70-4) and MS436 (molecular formula: C18H17N5O3S; CAS: 1395084-25-9) are two BET bromodomain inhibitors that effectively inhibit BRD4. Summary of the Invention

[0006] The purpose of this invention is to address the current situation where abnormal erythrocyte differentiation leads to disease, and the problem of low maturity of erythrocytes generated in vitro. This invention provides a method to promote terminal differentiation of erythrocytes.

[0007] This invention provides a method for promoting terminal differentiation of erythrocytes, achieved by adding a BRD4 inhibitor (+)-JQ1 or MS436 during the induction of terminal differentiation of erythrocytes. Specifically, it is implemented through the following steps.

[0008] 1. For the differentiation of umbilical cord blood-derived hematopoietic stem cells and adult-derived hematopoietic stem cells, mononuclear cells were first obtained using Ficoll density gradient centrifugation, and then CD34+ hematopoietic stem cells were sorted using a human CD34-positive cell sorting kit for downstream differentiation into erythrocytes.

[0009] 2. For the differentiation of peripheral blood-derived mononuclear cells, mononuclear cells obtained by Ficoll density gradient centrifugation are directly used for downstream differentiation.

[0010] 3. The erythrocyte differentiation process used in steps 1 and 2 above is as follows: (a) The first stage of differentiation, with 1-2 10 4 hematopoietic stem cells per cell / ml, or 4-6 10 6Mononuclear cells per cell / ml were seeded into the first-stage erythroid differentiation medium, and erythroid precursor cells were obtained after 7 days of differentiation. The components of the first-stage erythroid differentiation medium were: 100 ng / ml stem cell factor (SCF), 3 U / ml erythropoietin (EPO), 10 ng / ml interleukin-3 (IL-3), and 50 g / ml total transferrin, 40 ng / ml insulin-like growth factor-1 (IGF-1), 50 3-Isobutyl-1-methylxanthine (1-Methyl-3-Isobutylxanthine, IBMX), 1 Dexamethasone (DEX), 100 U / ml penicillin, and 100 μg / ml streptomycin; (b) In the second stage of differentiation, GPA+CD36+ primitive erythrocytes from the erythroid precursor cells in step (a) are sorted out, and then... 10 5 The cells were seeded at a density of [cell / ml] into the second-stage erythroid differentiation medium and differentiated for 5 days. The second-stage erythroid differentiation medium consisted of: 50 ng / ml SCF, 3 U / ml EPO, and 50 [units of other nutrients]. The procedure involves the addition of total transferrin (g / ml), penicillin (100U / ml), and streptomycin (100μg / ml), with the BRD4 inhibitor (+)-JQ1 or MS436.

[0011] The BRD4 inhibitor (+)-JQ1 is used at a concentration of 100 nM to 2 μM, and the BRD4 inhibitor MS436 is used at a concentration of 2 μM to 10 μM.

[0012] In step (b), BRD4 inhibitor (+)-JQ1 and MS436 are added and treated for 5 days during erythrocyte differentiation.

[0013] 4. Differentiation of the erythroid leukemia cell line TF-1: TF-1 cell line with 1 10 5 Cells / ml were cultured in maintenance medium for 3 days, centrifuged at 300 g for 5 min, washed once with PBS, resuspended, and counted. 10 5Differentiation was induced in the differentiation medium at a cell / ml density. BRD4 inhibitor (+)-JQ1 or MS436 was added during differentiation.

[0014] Maintenance medium components: RPMI 1640 basal medium, 10% fetal bovine serum, 2 ng / mL recombinant human granulocyte-macrophage colony-stimulating factor (GM-CSF).

[0015] Differentiation medium components: RPMI 1640 basal medium, 10% fetal bovine serum, 20 μM Hemin.

[0016] Another object of the present invention is to provide the application of the method in promoting terminal differentiation of erythrocytes, wherein the application is to induce erythrocyte differentiation using umbilical cord blood and adult-derived hematopoietic stem cells, peripheral blood mononuclear cells, and in the differentiation of the erythroid leukemia cell line TF-1.

[0017] This invention provides a simple method to promote erythrocyte differentiation by adding the BRD4 inhibitor (+)-JQ1 or MS436. This method is applicable to the differentiation process of erythrocytes from different cell sources and can improve the maturation of induced erythrocytes in vitro. This method can promote the differentiation of the erythroid leukemia cell line TF-1, providing a therapeutic strategy for erythroid-related diseases caused by abnormal erythrocyte differentiation. Attached Figure Description

[0018] Figure 1 (+)-JQ1 promotes the terminal differentiation of umbilical cord blood-derived hematopoietic stem cells into erythrocytes.

[0019] Figure 2 MS436 promotes the terminal differentiation of umbilical cord blood-derived hematopoietic stem cells into erythrocytes.

[0020] Figure 3 (+)-JQ1 and MS436 promote the terminal differentiation of adult-derived hematopoietic stem cells into erythrocytes.

[0021] Figure 4 (+)-JQ1 and MS436 promote the terminal differentiation of peripheral blood-derived mononuclear cells into erythrocytes.

[0022] Figure 5 (+)-JQ1 and MS436 promote the differentiation of the erythroid leukemia cell line TF-1. Detailed Implementation

[0023] The present invention will be described in detail with reference to the accompanying drawings and specific embodiments. The specific embodiments described herein are for illustration and explanation only and are not intended to limit the present invention.

[0024] Example 1: Umbilical cord blood-derived hematopoietic stem cells differentiate into erythrocytes.

[0025] Ficoll density gradient centrifugation for the separation of mononuclear cells from umbilical cord blood: (1) Dilute the umbilical cord blood to twice its volume with PBS (mix well with a Pasteur pipette); (2) Add Ficoll (Tianjin Haoyang Biological Products Technology Co., Ltd.) that has been brought to room temperature beforehand to the centrifuge tube. The volume of Ficoll should be half of the diluted blood sample in (1). (3) Slowly add the blood sample from (1) to the centrifuge tube containing Ficoll. The centrifuge tube can be tilted slightly, and the Pasteur pipette should be placed close to the Ficoll solution to slowly add the blood. When adding blood, be very gentle to avoid mixing the added blood with the Ficoll solution. This step is crucial. If the blood layer and the Ficoll solution layer mix, the PBMCs cannot be successfully separated. (4) Adjust the centrifuge parameters to A4, D0, 400 g, 25 min. This step is expected to take 45 min; (5) Remove the centrifuge tube. At this point, the liquid inside the centrifuge tube is divided into four layers. Mononuclear cells are mainly located in the upper layer of Ficoll solution, that is, the boundary between the plasma layer and the Ficoll solution layer (white membrane layer). The turbid flocculent material visible to the naked eye is the mononuclear cells. Carefully aspirate the white membrane layer with a pipette, trying to remove as much of the turbid flocculent material as possible. Aspirating a small amount of plasma will not affect the results, but you should avoid aspirating the red blood cells in the bottom layer. Note that you should handle the centrifuge tube gently at this time and do not remix the layered liquids. (6) Transfer the white film layer to a new centrifuge tube and dilute with PBS (3-5 times). 300 g, 10 min, A9, D9; (7) Resuspend the mononuclear cell pellet in PBS and count the cells.

[0026] CD34 positive selection kit sorts CD34-positive hematopoietic stem cells from single nuclei. (1) Adjust the concentration of mononuclear cells obtained in

[0024] to 5 × 10⁻⁶ cells using PBS containing 0.5% BSA. 7 / ml; (2) Transfer the cells from (1) to a 5ml sterile flow cytometer, add 50μL of the separation antibody combination (StemCell, Canada, Catalog #17856) per milliliter of sample and mix well. Incubate at room temperature for 5 min. (3) Shake RapidSpheres™ (StemCell, Canada, Catalog #17856) for 30 seconds. Add 40 μL of RapidSpheres™ to each milliliter of sample and mix well. Incubate at room temperature for 30 seconds. (4) Add a certain amount of PBS containing 0.5% BSA to a total volume of 2.5 mL; (5) Place the sterile flow cytometer containing the sample into the EasySep™ magnet (StemCell, Canada, Catalog #18000) and let it stand at room temperature for 3 minutes. (6) Pour out the magnetic poles and the sterile flow cytometry tubes connected to them to discard the supernatant in the sterile flow cytometry tubes. The cells adsorbed onto the tube wall by the magnetic poles are CD34-positive hematopoietic stem cells; (7) Remove the flow cytometer from the magnetic pole, resuspend it in 2.5 mL of PBS containing 0.5% BSA, and then discard the supernatant as in (6); (8) Repeat step (7) 2-3 times; (9) Remove the flow cytometer from the magnetic pole, add 2.5 mL of PBS containing 0.5% BSA to resuspend the cells, centrifuge at 300g for 5 min, and the cell pellet is CD34 positive hematopoietic stem cells.

[0027] Inducing CD34-positive hematopoietic stem cells to differentiate into erythrocytes.

[0028] Phase 1 of Differentiation: Directed Differentiation of the Red Lineage CD34-positive hematopoietic stem cells derived from umbilical cord blood were resuspended in the first-stage culture medium, counted, and quantified at 1-2... 10 4 CD34-positive hematopoietic stem cells were seeded into 12-well cell culture plates, with 1 ml of stage 1 culture medium added to each well. On day 4 of differentiation, 1 ml of culture medium was added again, and the cells were cultured for 7 days.

[0029] The first stage culture medium consisted of: 100 ng / ml SCF, 3 U / ml EPO, 10 ng / ml IL3, 50 μg / ml total transferrin, 40 ng / ml IGF1, 50 μM IBMX, 1 μM Dexamethasone, 100 U / ml penicillin, and 100 μg / ml streptomycin.

[0030] Flow cytometry sorting of GPA+CD36+ primitive red blood cells (1) Preparation of cell suspension: Cells differentiated to day 7 were placed in a flow cytometry tube, centrifuged at 300 g for 5 min, and the supernatant was discarded. After resuspending and washing with 2 ml PBS, the cells were centrifuged at 300 g for 5 min, and then rinsed with PBS at 2×10⁻⁶ ml. 7 Resuspended at a density of / ml; (2) Antibody labeling: at 1×10 7 Add 7 μl of GPA-APC and CD36-FITC flow cytometry antibody to each cell per ml of cells, and incubate at 4°C in the dark for 30 min. (3) Washing: Add 2 volumes of PBS to each tube, mix well, centrifuge at 300g for 5 min at room temperature, and discard the supernatant; (4) Resuspend the cells in flow cytometry cell sorting buffer (PBS containing 0.5% BSA and 2% penicillin and streptomycin) to prepare a cell concentration of 2-4 × 10⁻⁴. 7 / ml; (5) Under sterile conditions, select and collect GPA+CD36+ positive primitive red blood cells in flow cytometry cell sorting buffer. After sorting, take a small number of cells back for testing to determine whether the sorting purity is greater than 90%.

[0031] Second stage of differentiation: terminal differentiation of primitive erythrocytes The sorted GPA+CD36+ primitive erythrocytes were resuspended in the second-stage culture medium, counted, and divided into groups of 1. 10 5 Seed cells at a density of 1 / ml into 12-well cell culture plates, with 1 ml of Phase II medium added to each well. On day 10 of differentiation (day 3 of Phase II), 1 ml of medium was added, and on day 11 (day 4 of Phase II), 0.5 ml of medium was added, continuing until day 12 (day 5 of Phase II). Throughout the Phase II culture, 100 nM (+)-JQ1 or 8 μM MS436 was added to the medium.

[0032] The second-stage culture medium consisted of 50 ng / ml SCF, 3 U / ml EPO, 50 μg / ml total transferrin, 100 U / ml penicillin, and 100 μg / ml streptomycin.

[0033] Example 2: Differentiation of adult-derived hematopoietic stem cells into erythrocytes The method for terminal differentiation of adult hematopoietic stem cells into erythrocytes is the same as that for umbilical cord blood-derived hematopoietic stem cells into erythrocytes. The difference is that firstly, Ficoll density gradient centrifugation is used to obtain mononuclear cells from mobilized peripheral blood, and then CD34-positive hematopoietic stem cells are sorted using a human CD34-positive cell sorting kit (Stemcell, 17856) for downstream differentiation.

[0034] Example 3: Differentiation of peripheral blood-derived mononuclear cells into erythrocytes For the differentiation of peripheral blood mononuclear cells, the method for differentiating umbilical cord blood-derived hematopoietic stem cells into erythrocytes is similar. The difference lies in first obtaining peripheral blood mononuclear cells using Ficoll density gradient centrifugation, and then centrifuging at 4-6... 10 6 The density of / ml is used directly for downstream differentiation.

[0035] Example 4 Differentiation of the TF-1 erythroid leukemia cell line TF-1 cell line with 1 10 5 Cells / ml were cultured in maintenance medium for 3 days, centrifuged at 300 g for 5 min, resuspended in differentiation medium, and counted. 10 5 Differentiation was induced at a cell / ml density. During differentiation, the BRD4 inhibitor (+)-JQ1 or MS436 was added. The effects of BRD4 inhibitor (+)-JQ1 or MS436 on the differentiation of TF-1 cells into erythroid cells are as follows... Figure 5 As shown.

[0036] Maintenance medium composition: RPMI 1640 basal medium (Corning, USA), 10% serum, 2 ng / mL recombinant human granulocyte-macrophage colony-stimulating factor (GM-CSF).

[0037] Differentiation medium composition: RPMI 1640 basal medium, 10% serum, 20 μM Hemin.

[0038] Example 5: Detection of terminal differentiation of erythrocytes by flow cytometry (1) Preparation of cell suspension: For red blood cells at different differentiation stages to be tested, take 1×10 5 Add cells to a flow cytometry tube, resuspend and wash with 1 ml PBS, centrifuge at 300 g for 5 min, and discard the supernatant; (2) Antibody labeling: Resuspend the cells in 100 μL PBS to form a single-cell suspension, add 1 μL of the antibody to be detected for the surface antigen, and incubate at 4°C in the dark for 30 min; (3) Washing: Add 1 mL of PBS buffer to each tube, mix well, centrifuge at 300 g for 5 min at room temperature, and discard the supernatant; (4) Add 300-400 μL of PBS to the flow cytometer tube to resuspend the cells, and then perform flow cytometer analysis. (5) Data analysis using FlowJo X 10.0.7r2 software. The terminal differentiation of erythrocytes during the induction of peripheral blood mononuclear cell differentiation from umbilical cord blood-derived and mobilized peripheral blood hematopoietic stem cells is shown below. Figure 1 , Figure 2 , Figure 3 , Figure 4 As shown in the figure. These results indicate that the BRD4 inhibitors (+)-JQ1 or MS436 promote terminal differentiation of erythrocytes.

Claims

1. A method for promoting terminal differentiation of erythrocytes, characterized in that, This is achieved by adding the BRD4 inhibitor (+)-JQ1 or MS436 during the induction of terminal differentiation of erythrocytes, specifically through the following steps: (1) For the differentiation of umbilical cord blood-derived hematopoietic stem cells and adult-derived hematopoietic stem cells, mononuclear cells were first obtained by Ficoll density gradient centrifugation, and then CD34 positive selection kits were used to sort CD34 cells. + Hematopoietic stem cells are used for downstream differentiation into erythrocytes; (2) For the differentiation of peripheral blood mononuclear cells, mononuclear cells obtained by Ficoll density gradient centrifugation are directly used for downstream differentiation; (3) The cell differentiation process in steps (1) and (2) above is as follows: (a) The first stage of differentiation, 1-2 10 4 hematopoietic stem cells per cell / ml, or 4-6 10 6 Mononuclear cells per cell / ml were seeded into the first-stage erythroid differentiation medium and cultured for 7 days to obtain erythroid precursor cells. The first stage of erythroid differentiation culture medium consisted of: 100 ng / ml stem cell factor, 3 U / ml erythropoietin, 10 ng / ml interleukin-3, and 50... g / ml total transferrin, 40 ng / ml insulin-like growth factor-1, 50 3-Isobutyl-1-methylxanthine, 1 Dexamethasone, 100 U / ml penicillin, 100 μg / ml streptomycin; (b) The second stage of differentiation involves the transfer of GPA from the erythroid precursor cells in step (a) to the erythroid precursor cells. + CD36 + The primitive red blood cells were sorted out, with 1 10 5 The cells were seeded at a density of 1 cell / ml into the second-stage erythroid differentiation medium and differentiated for 5 days. The second-stage erythroid differentiation medium consisted of: 50 ng / ml SCF, 3 U / ml EPO, and 50... The process involves adding g / ml total transferrin, 100 U / ml penicillin, and 100 μg / ml streptomycin, along with the BRD4 inhibitor (+)-JQ1 or MS436. (4) Differentiation of the erythroid leukemia cell line TF-1 TF-1 cell line with 1 10 5 The cell / ml density was amplified in maintenance medium for 3 days, centrifuged at 300 g for 5 min, washed once with PBS, resuspended, and counted. 10 5 Differentiation was induced in the differentiation medium at a cell / ml density, with BRD4 inhibitor (+)-JQ1 or MS436 added during the differentiation process; Maintenance medium components: RPMI 1640 basal medium, 10% fetal bovine serum, 2 ng / mL recombinant human granulocyte-macrophage colony-stimulating factor; Differentiation medium components: RPMI 1640 basal medium, 10% fetal bovine serum, 20 Hemin.

2. The method according to claim 1, characterized in that, In steps (3) and (4), the concentration of BRD4 inhibitor (+)-JQ1 used is 100 nM to 2 μM, and the concentration of BRD4 inhibitor MS436 used is 2 μM to 10 μM.

3. The method according to claim 1, characterized in that, In step (3), BRD4 inhibitor (+)-JQ1 or MS436 is added to the treatment for 5 days during the erythrocyte differentiation process.

4. The method of claim 1 is used in promoting terminal differentiation of erythrocytes, characterized in that, The application involves using umbilical cord blood and adult-derived hematopoietic stem cells and peripheral blood mononuclear cells to induce erythrocyte differentiation, as well as in the differentiation process of the TF-1 erythroid leukemia cell line.

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