Application of thyroid hormone receptor beta agonist in reduction of erythrocyte apoptosis in in-vitro erythrocyte differentiation process

Abstract

The invention discloses application of a thyroid hormone receptor beta agonist in reduction of erythrocyte apoptosis in an in-vitro erythrocyte differentiation process, and finds that the thyroid hormone receptor beta agonist can reduce the quantity of erythrocyte apoptosis or death in the erythrocyte differentiation process for the first time. The problem of cell apoptosis or death in the final stage in the erythrocyte differentiation process in the field at present is solved. The research greatly promotes large-scale production and preparation of in-vitro red blood cells, and solves the problems of clinical blood shortage and safety.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and in particular, the present invention relates to the use of a thyroid hormone receptor β agonist in reducing erythrocyte apoptosis during in vitro erythrocyte differentiation. Background Art

[0002] Red blood cells are an important type of cell in the blood, mainly responsible for transporting oxygen from the lungs to tissues throughout the body and taking carbon dioxide from the tissues back to the lungs for excretion. It is the most abundant blood cell in the human body, accounting for 99% of the total number of cells in the blood. Red blood cell transfusion is an important clinical method for treating severe anemia and acute blood loss, and is widely used in the treatment of postoperative recovery, trauma, chronic anemia and many other diseases. At present, clinical blood use mainly relies on voluntary blood donations, but with the sharp increase in blood demand, blood supply shortages have become a major global public health problem. The global demand for blood is about 305 million units per year, but the global supply is only 272 million units. This supply shortage is particularly serious in low-income and middle-income countries. In addition, safety issues such as disease infection and transmission that may occur during blood transfusion have brought severe challenges to the safety of clinical blood use and seriously threatened human health. Therefore, how to obtain a safe, effective, sufficient and reliable source of blood has become a global problem that needs to be solved urgently in the current clinic.

[0003] In recent years, with the rapid development of stem cell research and regenerative medicine, new stem cell-based cell therapy has become the most promising application for solving major clinical diseases. At present, it is possible to induce hematopoietic stem cells from different sources to differentiate into mature red blood cells in vitro, but during the differentiation process, a large number of terminal red blood cells often undergo apoptosis and death, which seriously affects the production and quality of red blood cells and makes it difficult to meet the demand for clinical blood use.

[0004] Therefore, improving the apoptosis or death of terminal erythrocytes during in vitro erythrocyte differentiation becomes the key to achieving large-scale production of erythrocytes in vitro. Summary of the invention

[0005] In order to make up for the deficiencies of the prior art, the object of the present invention is to provide a method for reducing erythrocyte apoptosis during in vitro erythrocyte differentiation.

[0006] In order to achieve the above object, the present invention adopts the following technical solution:

[0007] The first aspect of the present invention provides the use of a thyroid hormone receptor β agonist in reducing erythrocyte apoptosis during in vitro erythrocyte differentiation.

[0008] Furthermore, the thyroid receptor β agonists include T3, T4, GC-1, and KB-141.

[0009] Furthermore, the thyroid receptor β agonist is T3 or GC-1.

[0010] Furthermore, the concentration of the T3 or GC-1 is 0.1 μM to 10 μM.

[0011] Furthermore, the concentration of T3 or GC-1 is 1 μM.

[0012] In some embodiments, the present invention has experimentally verified for the first time that the addition of a thyroid receptor β agonist during the in vitro differentiation of erythrocytes can significantly reduce the number of apoptotic erythrocytes.

[0013] A second aspect of the present invention provides a culture system for reducing erythrocyte apoptosis during erythrocyte differentiation in vitro, wherein the culture system comprises a thyroid receptor β agonist.

[0014] Furthermore, the thyroid receptor β agonists include T3, T4, GC-1, and KB-141.

[0015] Furthermore, the thyroid receptor β agonist is T3 or GC-1.

[0016] Furthermore, the concentration of the T3 or GC-1 is 0.1 μM to 10 μM.

[0017] Furthermore, the concentration of T3 or GC-1 is 1 μM.

[0018] Furthermore, the culture system also includes an erythrocyte essential culture medium, an erythrocyte expansion culture medium and an erythrocyte maturation culture medium.

[0019] Furthermore, the erythrocyte essential culture medium comprises basal differentiation culture medium, SCF, IL-3, EPO, and dexamethasone.

[0020] Furthermore, the red blood cell expansion medium comprises a basic differentiation medium, SCF, and EPO.

[0021] Furthermore, the erythrocyte maturation medium comprises a basal differentiation medium and EPO.

[0022] Furthermore, the basic differentiation medium contains IMDM, bovine serum albumin, ITS-X, ascorbic acid, acetylcysteine, and Trolox.

[0023] Furthermore, the contents of the components in the red blood cell essential culture medium are: 50 μg / mL SCF, 10 μg / mL IL-3, 20 μg / mL EPO, and 1 μM dexamethasone.

[0024] Furthermore, the contents of the components in the red blood cell expansion medium are: 50 μg / mL SCF, 20 μg / mL EPO.

[0025] Furthermore, the content of EPO in the erythrocyte maturation culture medium is 10 μg / mL.

[0026] Furthermore, the contents of the components in the basic differentiation medium are: 0.5% bovine serum albumin, 1% ITS-X, 50 μg / mL ascorbic acid, 50 μM acetylcysteine, and 50 μM Trolox.

[0027] In some embodiments, the specific numerical ranges or specific values ​​of each component and content in the culture medium listed in the present invention are only used to explain the technical effects achieved by the present invention and cannot be understood as a limitation on the present invention. Ordinary technicians in this field can make various adjustments or modifications based on the specific numerical ranges or specific values ​​listed in the present invention. As long as the expected inducing differentiation of hematopoietic stem cells or hematopoietic progenitor cells into red blood cells can be achieved, the specific data ranges or specific values ​​after adjustment or modification are also included in the protection scope of the present invention.

[0028] In the present invention, the term "erythrocyte" refers to an enucleated cell with characteristic markers of erythrocyte maturation, which specifically express glycoprotein A (CD235a) and do not express the marker CD36. Erythrocytes are the most numerous type of blood cells in the blood, and are also the main medium for transporting oxygen from the lungs or gills to various tissues of the body through the blood in vertebrates. The main functional molecule of erythrocytes is hemoglobin, which accounts for 90% of erythrocytes. Hemoglobin is a protein molecule containing heme, which can bind to oxygen molecules in the lungs or gills, and then release the bound oxygen molecules in the body's tissues. Oxygen molecules can easily diffuse through the cell membrane of erythrocytes. Hemoglobin can also transport carbon dioxide produced after the organism uses oxygen (less than 2% of the total amount of oxygen, and more carbon dioxide is transported by plasma). Another related protein molecule, myoglobin, can store oxygen in muscle cells.

[0029] In some embodiments, the in vitro differentiation process of erythrocytes consists of the following stages: (a) differentiation from hematopoietic stem / progenitor cells to erythroid progenitor cells; (b) differentiation of erythroid progenitor cells to proerythroblasts; (c) differentiation from proerythroblasts to proerythroblasts; (d) differentiation from proerythroblasts to intermediate erythroblasts; (e) differentiation from intermediate erythroblasts to metaerythroblasts; (f) differentiation from metaerythroblasts to reticulocytes; (g) differentiation from reticulocytes to erythrocytes. Among them, the term "erythroid progenitor cells" refers to a cell population between hematopoietic stem cells and erythroid precursor cells. The stage of differentiation from erythroid progenitor cells to erythroid precursor cells is a key process in regulating the self-stabilization mechanism of erythropoiesis. The term "erythroid precursor cells" refers to a cell population between erythroid progenitor cells and erythrocytes that is differentiated under the action of erythropoietin (EPO). The "erythroid progenitor cells" and "erythroid precursor cells" express CD36. In the present invention, adding the culture system comprising the thyroid receptor β agonist described in the second aspect of the present invention to any process of in vitro differentiation of erythrocytes can reduce the apoptosis or death of cells occurring in the terminal stage of erythrocyte differentiation.

[0030] A third aspect of the present invention provides a method for reducing erythrocyte apoptosis during erythrocyte differentiation in vitro.

[0031] Furthermore, the method comprises culturing hematopoietic stem / progenitor cells using the culture system described in the second aspect of the present invention.

[0032] Further, the method comprises the following steps:

[0033] 1) Day 0-12, culturing hematopoietic stem / progenitor cells using the red blood cell essential medium described in the second aspect of the present invention;

[0034] 2) Day 12-15, culturing the cells obtained in step 1) using the red blood cell expansion medium described in the second aspect of the present invention;

[0035] 3) Day 15-35, culturing the cells obtained in step 2) using the red blood cell maturation medium described in the second aspect of the present invention;

[0036] 4) adding a thyroid receptor β agonist to the culture system.

[0037] Furthermore, the step 4) is performed on Day 0-9, Day 0-21, Day 6-21 or Day 9-21.

[0038] Furthermore, the thyroid receptor β agonists include T3, T4, GC-1, and KB-141.

[0039] Furthermore, the thyroid receptor β agonist is T3 or GC-1.

[0040] Furthermore, the concentration of the T3 or GC-1 is 0.1 μM to 10 μM.

[0041] Furthermore, the concentration of T3 or GC-1 is 1 μM.

[0042] Furthermore, the hematopoietic stem / progenitor cells include: hematopoietic stem / progenitor cells derived from umbilical cord blood, hematopoietic stem / progenitor cells derived from induced pluripotent stem cells, hematopoietic stem / progenitor cells derived from bone marrow, and hematopoietic stem / progenitor cells derived from peripheral blood.

[0043] Furthermore, the hematopoietic stem / progenitor cells are hematopoietic stem / progenitor cells derived from umbilical cord blood or hematopoietic stem / progenitor cells derived from induced pluripotent stem cells.

[0044] Furthermore, the cell density in step 1) is 1×10 5 Pieces / mL.

[0045] Furthermore, the cell density in step 2) is 5×10 5 cells / mL.

[0046] Furthermore, the cell density in step 3) is 1×10 6 Pieces / mL.

[0047] In some embodiments, the present invention demonstrates through experiments that during the in vitro differentiation of hematopoietic stem / progenitor cells into erythrocytes, adding a thyroid receptor β agonist to the culture system can reduce the number of terminal erythrocyte apoptosis and death.

[0048] In some embodiments, the present invention has experimentally verified that adding a thyroid receptor β agonist to the culture medium used during Day 0-9, Day 0-21, Day 6-21 or Day 9-21 can significantly reduce the number of erythrocyte apoptosis or death during erythrocyte differentiation in vitro, and the earlier the action time and the longer the action time, the fewer the number of erythrocyte apoptosis or death, that is, the thyroid receptor β agonist is beneficial to improving the quality and quantity of erythrocytes and reducing the number of apoptosis and death.

[0049] In the present invention, the term "hematopoietic stem / progenitor cells" refers to hematopoietic stem cells (HSC) and / or hematopoietic progenitor cells (HPC), wherein the term "hematopoietic stem cells" refers to stem cells that can produce all blood cell types of the three hematopoietic lineages (erythroid, lymphoid and myeloid), including myeloid lineage (monocytes and macrophages, neutrophils, basophils, eosinophils, erythrocytes, megakaryocytes / platelets, dendritic cells) and lymphoid lineage (T cells, B cells, NK cells). The term "hematopoietic progenitor cell" refers to a hematopoietic stem cell that proliferates and differentiates into progenitor cells of various blood cells under the regulation of a certain microenvironment and certain factors. It is also a rather primitive cell with the ability to proliferate, but has lost its multidirectional differentiation ability and can only proliferate and differentiate toward one or several blood cell lines, so it is also called a committed stem cell. Hematopoietic progenitor cells can differentiate into multipotent cells of several cell types of the hematopoietic system, including but not limited to: granulocytes, monocytes, erythrocytes, megakaryocytes, B cells and T cells. Hematopoietic progenitor cells are committed to the hematopoietic cell lineage and generally do not self-regenerate. The term "hematopoietic progenitor cell" includes short-term hematopoietic stem cells (ST-HSC), multipotent progenitor cells (MPP), common myeloid progenitor cells (CMP), granulocyte-monocyte progenitor cells (GMP), and megakaryocyte-erythrocyte progenitor cells (MEP). Hematopoietic stem / progenitor cells express CD45.

[0050] In some embodiments, the "hematopoietic stem / progenitor cells" may be obtained from any one or more of the following sources: embryonic tissue, umbilical cord blood, bone marrow, peripheral blood, circulating peripheral blood, stem cell lines, or may be obtained in vitro from other cells, such as embryonic stem cells, induced pluripotent stem cells (iPS cells) or adult pluripotent cells. The cells from the above sources may be amplified in vitro by any method acceptable to a person skilled in the art before use. In some cases, hematopoietic stem cells may be separated from any of the above sources (e.g., bone marrow) or cultured in vitro. If the cells used are derived from an immortal stem cell line, it may further facilitate easy acquisition and preparation of a sufficient number of cells. Hematopoietic stem cells or hematopoietic progenitor cells from various sources are within the scope of the present invention. In a specific embodiment of the present invention, the hematopoietic stem / progenitor cells are hematopoietic stem / progenitor cells derived from umbilical cord blood or hematopoietic stem / progenitor cells derived from induced pluripotent stem cells.

[0051] In the present invention, the term "induced pluripotent stem cells (iPSC)" refers to pluripotent stem cells obtained by genetic reprogramming of differentiated somatic cells and having a morphology partially similar to embryonic stem cells and the potential for self-renewal and pluripotency. These cells are particularly positive for pluripotency markers, including alkaline phosphatase staining and expression of proteins NANOG, SOX2, OCT4 and SSEA3 / 4. Methods for obtaining induced pluripotent stem cells are well known to those skilled in the art.

[0052] In some embodiments, when preparing the "induced pluripotent stem cells", various reagents for preparing induced pluripotent stem cells, such as reprogramming vectors, expression cassettes, culture media, etc., and even commercial induced pluripotent stem cells can be purchased from the market. hiPSC refers to induced pluripotent stem cells obtained by induction from human cells. In a specific embodiment of the present invention, the hiPSC used in the examples is prepared according to the preparation method disclosed in Chinese patent CN113462638A, and the entire text of the patent document is incorporated herein by reference.

[0053] In addition, the present invention also provides a method for preventing, treating and / or improving red blood cell-related diseases or disorders, the method comprising administering red blood cells induced to differentiate based on the method described in the second aspect of the present invention to a subject in need.

[0054] Furthermore, the red blood cell-related diseases or disorders refer to related diseases or disorders that require red blood cell transfusion, including but not limited to: severe anemia, acute blood loss, cancer, transplantation, autoimmune diseases, infectious diseases, inflammation, immunodeficiency-related diseases, etc.

[0055] In some embodiments, red blood cells are administered to the subject by one or more routes selected from systemic, topical, intravenous, subcutaneous, intraarticular, intramuscular, intrathecal, and intraperitoneal. In some embodiments, the subject includes one or more animals, including, for example, cattle, horses, sheep, primates, avian, and rodent species. The subject can be an animal (e.g., a mammal, bird, fish, reptile, or amphibian) whose blood contains red blood cells. In some embodiments, the subject can be a mammal, such as a human or non-human mammal. In other embodiments, the subject can be a mouse, rat, hamster, ferret, gerbil, rabbit, monkey, chimpanzee, horse, pony, donkey, sheep, pig, chicken, goat, cat, or dog. In a preferred embodiment, the subject is human.

[0056] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0057] The present invention discovered for the first time that thyroid receptor β agonists can reduce the number of erythrocyte apoptosis or death during erythrocyte differentiation, solving the problem of apoptosis or death in the terminal stage of erythrocyte differentiation in the current field. This research will greatly promote the large-scale production and preparation of in vitro erythrocytes and solve the problem of blood shortage and safety for clinical use. BRIEF DESCRIPTION OF THE DRAWINGS

[0058] Figure 1 The results of the serum-free culture system on in vitro erythrocyte differentiation are shown in Figure A; Figure A is a cell flow cytometry analysis of the effect of the serum-free culture system on the expression of erythrocyte markers CD36 and CD235a during erythrocyte differentiation; Figure B is a cell flow cytometry analysis of the effect of the serum-free culture system on the expression of erythrocyte markers CD71 and CD235a during erythrocyte differentiation;

[0059] Figure 2 The figure shows the results of cell flow cytometry analysis of cell apoptosis and death at different differentiation time points (Day0 - Day24) of in vitro erythroid differentiation;

[0060] Figure 3 Shows the chemical structure of T3 (Triiodothyronine);

[0061] Figure 4 Shows the chemical structure of GC-1 (Sobetirome);

[0062] Figure 5 Showing CD34 before and after magnetic bead sorting + HPSC ratio result diagram;

[0063] Figure 6 A graph showing the effects of thyroid hormone receptor β agonists T3 and GC-1 on red blood cell morphology;

[0064] Figure 7 The results of the effects of thyroid hormone receptor β agonists T3 and GC-1 on erythrocyte apoptosis or death are shown; Figure A is a flow cytometry analysis of the effects of thyroid hormone receptor β agonists T3 and GC-1 on erythrocyte apoptosis or death at different differentiation time points; Figure B is a flow cytometry analysis of the distribution of FSC and SSC of cells on day 21;

[0065] Figure 8The results of the effects of different action time windows and concentrations of thyroid hormone receptor β agonists T3 and GC-1 on erythrocyte apoptosis or death are shown in the figure; Figure A shows the effects of thyroid hormone receptor β agonists T3 and GC-1 on erythrocyte apoptosis or death on day 0-9 (D0-9), day 0-21 (D0-21) and day 9-21 (D9-21) on differentiation day 21, using flow cytometry analysis; Figure B shows the effects of different concentrations of thyroid hormone receptor β agonists T3 and GC-1 (0.1µM, 1µM and 10µM) on erythrocyte apoptosis or death on differentiation day 21, using flow cytometry analysis;

[0066] Fig. 9 The figure shows the results of microscopic imaging analysis of the effects of thyroid hormone receptor β agonists T3 and GC-1 on the morphology of red blood cells at different differentiation time points;

[0067] Fig.10 The graph shows the results of flow cytometry analysis of the effect of thyroid hormone receptor β agonist T3 and GC-1 on apoptosis or death of erythrocytes treated on days 6-21 at day 21 of differentiation. DETAILED DESCRIPTION

[0068] The present invention is further described below in conjunction with specific embodiments, which are only used to explain the present invention and are not to be construed as limiting the present invention. It will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to these embodiments without departing from the principles and purposes of the present invention, and that the scope of the present invention is defined by the claims and their equivalents. The experimental methods for which specific conditions are not specified in the following examples are usually tested under conventional conditions or under conditions recommended by the manufacturer.

[0069] The experimental materials used in the embodiments of the present invention are shown in Table 1 below.

[0070] Table 1 Experimental materials

[0071] Example 1 Preparation of red blood cells

[0072] 1. Preparation of hiPSC-derived HSPCs

[0073] hiPSCs were cultured at 8 × 10 4 cells / cm 2The cells were replated at a density of 10 μg / mL and maintained in E8 medium for 2 days. To induce HSPC differentiation, the medium was changed to STEMdiff™ APEL™ 2 medium (Stemcell, 05275) and 9 μM CHIR-99021 (Selleck, S1263) was added for 24 hours. Then, the medium was changed and 20 ng / mL VEGF and 20 ng / mL bFGF were added for 48 hours. Then, the cells were cultured at 2 × 10 5 cells / cm 2 The cells were replated at a density of 1.50 μg / mL and cultured with 50 ng / mL SCF, 30 ng / mL TPO, 20 ng / mL VEGF, 20 ng / mL bFGF, 10 ng / mL BMP4, 10 ng / mL IL3 and 10 ng / mL Flt-3L for 7 days, and then the suspended cells were collected to obtain hiPSC-derived HSPCs. Unless otherwise specified, all cytokines were purchased from Peprotech.

[0074] 2. Preparation of HSPCs from cord blood

[0075] 1) Take a 50 mL centrifuge tube and add an equal amount of separation solution (TBD Science, LTS1077) to the blood sample, see Table 2 for details;

[0076] 2) Carefully draw the blood sample with a pipette and add it to the surface of the separation solution. Centrifuge at 460 g for 40 min.

[0077] 3) After centrifugation, the centrifuge tube is divided into four layers from top to bottom, namely plasma layer, ring-shaped milky white mononuclear cell layer, transparent separation fluid layer, and red blood cell layer;

[0078] 4) Carefully pipette the milky white mononuclear cell layer into a new 50 mL centrifuge tube, add 5 times the volume of DPBS to the resulting centrifuge tube, mix the cells; centrifuge at 380 g for 10 min;

[0079] 5) Discard the supernatant and add appropriate amount of DPBS to resuspend the cells; centrifuge at 300 g for 10 min;

[0080] 6) After discarding the supernatant, resuspend the cells in the corresponding liquid according to the experiment. Then separate the CD34 according to the instructions of CD34 MicroBead Kit (Miltenyi Biotec, 130-046-703). + Cells, namely, HSPCs derived from cord blood are obtained.

[0081] Table 2 Relationship between the volume of blood to be separated, the volume of separation fluid, and the volume of the centrifuge tube used

[0082]

[0083] 3. Preparation of red blood cells by inducing differentiation of hiPSC-derived HSPCs

[0084] Serum contains a variety of components that are beneficial to cell culture and differentiation, such as growth factors, hormones, vitamins, minerals, amino acids, fatty acids, carbohydrates and proteins. These components provide cells with essential nutrients and growth signals, and promote cell proliferation, differentiation and survival. Commonly used serum includes fetal bovine serum (FBS), which provides cells with a variety of substances that support cell growth and maintenance. Therefore, serum is also used for in vitro differentiation and culture of red blood cells, and promotes cell survival to a certain extent. However, serum components are complex and the source is unclear. It may contain pathogenic microorganisms, viruses or other harmful substances, increasing the risk of infection. In addition, animal-derived proteins in serum may trigger immune responses, leading to adverse reactions in clinical patients. Therefore, reducing or replacing the use of serum can help significantly improve the safety and controllability of cell therapy.

[0085] In order to reduce the potential impact of serum on the clinical application of cell products, we have established a serum-free red blood cell differentiation process system. The details are as follows:

[0086] Supplements were gradually added to the basal medium to induce HSPC differentiation into the erythroid lineage at specific stages. The basal differentiation medium included IMDM (Stemcell, 05992), 0.5% bovine serum albumin (Proliant, 68700), 0.1% ITS-X (Gibco, 51500-056), 50 μg / mL ascorbic acid (Sigma, A8960), 50 μM acetylcysteine ​​(Selleck, S1623), and 50 μM Trolox (Sigma, 648471).

[0087] In the first step (days 0-12), HSPCs (1 × 10 5 Cells were cultured in erythroid essential medium (ESM) containing basal differentiation medium, 50 μg / mL SCF, 10 μg / mL IL-3, 20 μg / mL EPO, and 1 μM dexamethasone), and the medium was changed every 3 days.

[0088] In the second step (days 12-15), cells (5 × 10 5cells / mL) were resuspended in erythroid expansion medium (erythroid expansion medium contains: basal differentiation medium, 50 μg / mL SCF and 20 μg / mL EPO).

[0089] In the third step (days 15-35), cells (1 × 10 6 Cells were resuspended in erythroid maturation medium (containing basal differentiation medium and 10 μg / mL EPO). The medium was changed every 3 days and the cells were placed at 37°C and 5% CO 2 cultured in a cell culture incubator.

[0090] 4. Flow cytometry

[0091] To analyze cell surface markers, cells were incubated with directly labeled antibodies for 30 min at room temperature: CD71-FITC (Biolegend, 334104), CD36-PE (Biolegend, 336206), CD235a-APC (eBioscience, 17-9987-42). Isotope-matched antibodies were used as controls. Cell apoptosis and cell death were detected using the Annexin V-FITC / PI apoptosis detection kit, and the operating steps were referred to the instructions (Yeasen, 40302ES60). Flow cytometry data were acquired using the GuavaEasyCyte HT system (Luminex) and analyzed using FlowJo software (Tree Star).

[0092] 5. Experimental results

[0093] In order to study whether the serum-free culture system can effectively support the differentiation of erythrocytes in vitro, we detected the changes in the expression of erythrocyte markers CD36, CD71 and CD235a at different differentiation time points. The results of flow cytometry analysis showed that the serum-free culture system can effectively promote the expression of erythrocyte markers CD36, CD71 and CD235a, and CD36 + CD235a + and CD71 + CD235a + The number of erythroid progenitor cells gradually increases with the differentiation process, then gradually decreases at the terminal stage of differentiation and transforms into CD36 - CD235a + and CD71 - CD235a + of mature red blood cells ( Figure 1 ).

[0094] In order to study the effect of serum-free culture system on cell survival during differentiation, we used Annexin V-FITC / PI to detect changes in erythrocyte apoptosis and death at different differentiation time points. Flow cytometry results showed that compared with the early stages of differentiation (Day 0, Day 6, Day 9, Day 12, and Day 15), Annexin V-FITC / PI in cells at the late stages of differentiation (Day 18, Day 21, and Day 24) was significantly higher than that in cells at the early stages of differentiation (Day 0, Day 6, Day 9, Day 12, and Day 15). + PI - Early apoptotic cells and Annexin V-FITC + PI + The proportion of late apoptotic cells increased significantly ( Figure 2 ). The above results indicate that in the serum-free culture system, erythrocytes at the terminal stage of differentiation undergo severe apoptosis and death.

[0095] Example 2 Effects of thyroid hormone receptor β agonists T3 and GC-1 on apoptosis of terminal erythroid cells differentiated from umbilical cord blood-derived HSPCs

[0096] To explore the optimization of erythroid differentiation protocol by using thyroid hormone receptor β agonists T3 (Triiodothyronine, Selleck, S5726) and GC-1, the addition phase and concentration of T3 and GC-1 were studied, as well as DMSO as a control. Unless otherwise stated, all cytokines were purchased from Peprotech. The chemical structures of T3 and GC-1 are detailed in Figure 3 and Figure 4 .

[0097] The experimental groups were as follows: Control group—blank control group (treatment method was the same as in Example 1), T3 group (1 µM T3 was added during Day 0-21 of HSPC differentiation into erythrocytes in Example 1), GC-1 group (1 µM GC-1 was added during Day 0-21 of HSPC differentiation into erythrocytes in Example 1). Flow cytometry was performed on the 21st day of differentiation.

[0098] To ensure the purity of HSCP cells from cord blood, we used magnetic beads to enrich CD34 + HSPC cells. Flow cytometry results showed that the sorted CD34 + The proportion of HSPC cells reached 89.54%, while the CD34 + The proportion of HSPC cells is only 2.11%, see Figure 5 After sorting CD34 + HSPC cells with a proportion greater than 80% can be used for subsequent differentiation experiments.

[0099] Subsequently, we investigated whether the thyroid hormone receptor β agonists T3 and GC-1 affect the survival of terminal erythrocytes. We observed the morphological changes of cells on the 11th and 21st days of erythrocyte differentiation. The cell image results showed that on the 11th day of differentiation, the cells in the control group (Control) and the experimental group (T3 and GC-1) maintained complete cell morphology, exhibited good cell activity, and the cells were spherical; on the 21st day of differentiation, the control group (Control) had a large number of cell fragments and cells with incomplete structures, while the experimental group (T3 and GC-1) had only very few cell fragments, most of the cells maintained a complete cell structure, and the cells showed the morphological characteristics of biconcave mature erythrocytes, see for details. Figure 6 .

[0100] In addition, we used Annexin V-FITC / PI to detect changes in erythrocyte apoptosis and death at different differentiation time points. The flow cytometry results showed that on the 16th day of differentiation, Annexin V-FITC / PI in the control group (Control) + PI - The proportion of early apoptotic cells is 30.7%, Annexin V-FITC + PI + The proportion of late apoptotic cells was 9.93%; Annexin V-FITC + PI - The proportions of early apoptotic cells were 9.96% and 13.5%, respectively, and Annexin V-FITC + PI + The proportions of late apoptotic cells were 2.58% and 4.38% respectively; on the 21st day of differentiation, the Annexin V-FITC + PI - The proportion of early apoptotic cells is 24.7%, Annexin V-FITC + PI + The proportion of late apoptotic cells was 7.51%; Annexin V-FITC + PI - The proportions of early apoptotic cells were 9.12% and 16.7%, respectively, and Annexin V-FITC + PI + The cell proportions of late apoptotic cells were 3.19% and 5.48%, respectively. Figure 7 A. In addition, FSC and SSC flow cytometry analysis also showed that the cell debris in the T3 and GC-1 experimental groups was significantly reduced compared with the control group, further reflecting that there was less cell death or apoptosis. For details, see Figure 7B. The above experimental results show that the addition of thyroid hormone receptor β agonist to serum-free culture medium can effectively reduce apoptosis and death of cells in the terminal stage of erythroid differentiation.

[0101] In addition, to further analyze the effects of different time windows of action of thyroid hormone receptor β agonist T3 or GC-1 on erythrocyte apoptosis and death, we treated T3 1µM or GC-1 1µM on days 0-9 (D0-9), 0-21 (D0-21), and 9-21 (D9-21) of differentiation (T3 or GC-1 was added to the culture medium every day during the addition phase). The flow cytometry results on day 21 showed that T3 and GC-1 at different treatment times could reduce Annexin V-FITC + PI - Early apoptotic cells and Annexin V-FITC + PI + The proportion of late apoptotic cells, including Annexin V-FITC in T3 and GC-1 experimental groups treated on days 0-21 (D0-21) + PI - Early apoptotic cells and Annexin V-FITC + PI + The proportion of late apoptotic cells was the lowest, indicating that this period of time can effectively reduce the death and apoptosis of red blood cells ( Figure 8 A).

[0102] In order to obtain the optimal concentration of thyroid hormone receptor β agonist T3 or GC-1, we treated 0.1-10 µM T3 or GC-1 on days 0-21 of differentiation. The flow cytometry results on day 21 showed that different concentrations of T3 and GC-1 could reduce the expression of Annexin V-FITC + PI - Early apoptotic cells and Annexin V-FITC + PI + The proportion of late apoptotic cells, including Annexin V-FITC in the 1 µM T3 and 1 µM GC-1 experimental groups + PI - Early apoptotic cells and Annexin V-FITC + PI + The proportion of late apoptotic cells was the lowest, indicating that this concentration can effectively reduce the death and apoptosis of red blood cells ( Figure 8 B).

[0103] Example 3 Effects of thyroid hormone receptor β agonists T3 and GC-1 on apoptosis of terminal erythroid cells differentiated from hiPSC-derived HSPCs

[0104] We further analyzed whether the thyroid hormone receptor β agonist T3 or GC-1 also reduced apoptosis or death of erythrocytes differentiated from hiPSC-derived HSPCs. We treated the thyroid hormone receptor β agonist T3 (1 µM) or GC-1 (1 µM) on days 0-21 (D0-21), days 6-21 (D6-21), and days 9-21 (D9-21), respectively. The microscopic images on day 21 showed that compared with the control group, the addition of thyroid hormone receptor β agonist T3 or GC-1 at different time points effectively reduced cell apoptosis and death, and the treatment of T3 or GC-1 on D6-21 and D9-21 significantly increased the number of erythrocytes, see for details. Fig. 9 To further detect cell apoptosis and cell death, we selected D6-21 as the time window for hiPSC-derived HSPC differentiation into erythroid cells treated with thyroid hormone receptor β agonist T3 or GC-1. Flow cytometry results showed that on the 21st day of differentiation, Annexin V-FITC + PI - The proportion of early apoptotic cells is 14.54%, Annexin V-FITC + PI + The proportion of late apoptotic cells was 8.16%; Annexin V-FITC + PI - The proportions of early apoptotic cells were 8.62% and 6.64%, respectively, and Annexin V-FITC + PI + The cell proportions of late apoptotic cells were 4.25% and 3.13%, respectively. Fig.10 .

[0105] The above experimental results show that thyroid hormone receptor β agonists T3 and GC-1 can effectively reduce cell apoptosis or death during the differentiation of hiPSC-derived HSPCs into red blood cells.

[0106] The description of the above embodiments is only used to understand the method and core idea of ​​the present invention. It should be pointed out that, for those skilled in the art, several improvements and modifications can be made to the present invention without departing from the principle of the present invention, and these improvements and modifications will also fall within the scope of protection of the claims of the present invention.

Claims

1. The use of thyroid hormone receptor β agonists in reducing erythrocyte apoptosis during in vitro erythrocyte differentiation; Preferably, the thyroid receptor β agonist includes T3, T4, GC-1, KB-141; Preferably, the thyroid receptor β agonist is T3 or GC-1; Preferably, the concentration of T3 or GC-1 is 0.1 μM to 10 μM; More preferably, the concentration of T3 or GC-1 is 1 μM.

2. A culture system for reducing erythrocyte apoptosis during erythrocyte differentiation in vitro, characterized in that: The culture system contains a thyroid receptor beta agonist.

3. The culture system according to claim 2, characterized in that: The thyroid receptor β agonists include T3, T4, GC-1, and KB-141; Preferably, the thyroid receptor β agonist is T3 or GC-1; Preferably, the concentration of T3 or GC-1 is 0.1 μM to 10 μM; More preferably, the concentration of T3 or GC-1 is 1 μM.

4. The culture system according to claim 2, characterized in that: The culture system also includes an erythrocyte essential medium, an erythrocyte expansion medium, and an erythrocyte maturation medium; The erythrocyte essential medium comprises a basic differentiation medium, SCF, IL-3, EPO, and dexamethasone; The red blood cell expansion medium comprises a basic differentiation medium, SCF, and EPO; The erythrocyte maturation medium comprises a basal differentiation medium and EPO; The basic differentiation medium contains IMDM, bovine serum albumin, ITS-X, ascorbic acid, acetylcysteine, and Trolox.

5. The culture system according to claim 4, characterized in that: The contents of the components in the red blood cell essential medium are: 50 μg / mL SCF, 10 μg / mL IL-3, 20 μg / mL EPO, and 1 μM dexamethasone; Preferably, the contents of the components in the red blood cell expansion medium are: 50 μg / mL SCF, 20 μg / mL EPO; Preferably, the content of EPO in the erythrocyte maturation medium is 10 μg / mL; Preferably, the contents of the components in the basic differentiation medium are: 0.5% bovine serum albumin, 1% ITS-X, 50 μg / mL ascorbic acid, 50 μM acetylcysteine, and 50 μM Trolox.

6. A method for reducing erythrocyte apoptosis during erythrocyte differentiation in vitro, characterized in that: The method comprises culturing hematopoietic stem / progenitor cells using the culture system described in any one of claims 2 to 5.

7. The method according to claim 6, characterized in that The method comprises the following steps: 1) Day 0-12, culturing hematopoietic stem / progenitor cells using the red blood cell essential medium described in claim 3 or 4; 2) Day 12-15, culturing the cells obtained in step 1) using the red blood cell expansion medium described in claim 3 or 4; 3) Day 15-35, culturing the cells obtained in step 2) using the erythrocyte maturation medium described in claim 3 or 4; 4) adding a thyroid receptor β agonist to the culture system.

8. The method according to claim 6 or 7, characterized in that: The step 4) is performed on Day 0-9, Day 0-21, Day 6-21 or Day 9-21; Preferably, the thyroid receptor β agonist includes T3, T4, GC-1, KB-141; Preferably, the thyroid receptor β agonist is T3 or GC-1; Preferably, the concentration of T3 or GC-1 is 0.1 μM to 10 μM; More preferably, the concentration of T3 or GC-1 is 1 μM.

9. The method according to claim 6, characterized in that The hematopoietic stem / progenitor cells include: hematopoietic stem / progenitor cells derived from umbilical cord blood, hematopoietic stem / progenitor cells derived from induced pluripotent stem cells, hematopoietic stem / progenitor cells derived from bone marrow, and hematopoietic stem / progenitor cells derived from peripheral blood; Preferably, the hematopoietic stem / progenitor cells are hematopoietic stem / progenitor cells derived from umbilical cord blood or hematopoietic stem / progenitor cells derived from induced pluripotent stem cells.

10. The method according to claim 7, characterized in that The cell density in step 1) is 1×10 5 Pieces / mL; Preferably, the cell density in step 2) is 5×10 5 cells / mL; Preferably, the cell density in step 3) is 1×10 6 Pieces / mL.

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