Application of ULK1 inhibitor in promotion of erythroid progenitor cell amplification

Abstract

The invention provides application of a ULK1 inhibitor in promoting erythroid progenitor cell amplification, and relates to the technical field of biology. The application comprises: applying the ULK1 inhibitor to (I) to promote erythroid progenitor cell amplification; (2) preparing a product for amplifying erythroid progenitor cells; (3) in-vitro induced differentiation of red blood cells; (IV) preparing a product for in-vitro induced differentiation of red blood cells; and (V) preparing a blood transfusion product. The ULK1 inhibitor has the effects of promoting red blood cell amplification and improving in-vitro induced differentiation of red blood cells.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and particularly to the use of a ULK1 inhibitor in promoting the expansion of erythroid progenitor cells. Background Art

[0002] Red blood cells are the most numerous blood cells in mammals, performing physiological functions such as oxygen carrying and immune regulation. Clinically, red blood cells are mainly used for the treatment of various diseases such as post-operative and trauma, severe anemia, and malignant blood diseases. Obtaining red blood cells in vitro is one of the ways to solve the shortage of clinical blood supply.

[0003] Currently, the in vitro induction and differentiation of red blood cells are mainly divided into three stages: The first stage is the directional induction and differentiation of hematopoietic stem and progenitor cells from different sources (including human pluripotent stem cells, cord blood, and peripheral blood) into erythroid progenitor cells, which takes 7 days; the second stage is to induce the further differentiation of erythroid progenitor cells into erythroid precursor cells (including primitive erythrocytes, proerythroblasts, erythrocytes in the middle stage, and erythrocytes in the late stage); the third stage is to induce the enucleation of late erythrocytes to form reticulocytes. The entire erythroid induction and differentiation process takes about 20 days.

[0004] In recent years, multiple laboratories have successively achieved the in vitro acquisition of red blood cells from different sources, such as cord blood, mobilized peripheral blood, human pluripotent stem cells, and immortalized erythroid cell lines. However, these in vitro-generated red blood cells all have the problem of low amplification efficiency and cannot meet the clinical routine dosage. The large production of red blood cells mainly depends on the number of erythroid progenitor cells. Promoting the expansion of erythroid progenitor cells is beneficial to achieving the purpose of increasing production. Therefore, how to promote the expansion of erythroid progenitor cells is a problem to be solved at present.

[0005] In view of this, the present invention is specifically proposed. Summary of the Invention

[0006] The purpose of the present invention is to provide the use of a ULK1 inhibitor in promoting the expansion of erythroid progenitor cells to alleviate the problem of low amplification efficiency of in vitro-generated red blood cells.

[0007] To solve the above technical problems, the present invention specifically adopts the following technical solutions: In the first aspect, there is provided the use of a ULK1 inhibitor in any one of (I) to (V): (I) Promoting the expansion of erythroid progenitor cells; (II) Preparing a product for the expansion of erythroid progenitor cells; (III) In vitro induction and differentiation of red blood cells; (IV) Preparing a product for the in vitro induction and differentiation of red blood cells; (V) Preparing a blood transfusion product.

[0008] In a second aspect, a composition for erythrocyte preparation is provided, the composition comprising a first-stage culture composition, the first-stage culture composition comprising a ULK1 inhibitor, stem cell factor, erythropoietin, interleukin-3, holotransferrin, insulin-like growth factor-1, 3-isobutyl-1-methylxanthine, and dexamethasone.

[0009] In a third aspect, a culture medium for erythrocyte preparation is provided, the culture medium comprising the composition according to the second aspect and a basal medium.

[0010] In a fourth aspect, a method for differentiating hematopoietic stem and progenitor cells into erythroid progenitor cells is provided, the method comprising culturing hematopoietic stem and progenitor cells using a differentiation medium containing a ULK1 inhibitor to differentiate the hematopoietic stem and progenitor cells into erythroid progenitor cells.

[0011] In a fifth aspect, a method for in vitro induced differentiation of erythrocytes is provided, the method comprising obtaining erythroid progenitor cells using the method according to the fourth aspect, and then culturing the erythroid progenitor cells in a differentiation induction medium without a ULK1 inhibitor.

[0012] Compared with the prior art, the present invention has the following beneficial effects: The present invention discovers that a ULK1 inhibitor can promote the expansion of erythroid progenitor cells. Adding an appropriate concentration of the ULK1 inhibitor SBI-0206965 to the medium for differentiating hematopoietic stem and progenitor cells into erythroid progenitor cells can promote the differentiation of hematopoietic stem and progenitor cells into erythroid progenitor cells without affecting cell viability, and the ability of erythroid progenitor cells to produce erythrocytes is enhanced. Applying the ULK1 inhibitor to promote the expansion of erythroid progenitor cells, in vitro induced differentiation of erythrocytes, and preparation of related products can improve the efficiency of obtaining erythrocytes in vitro, reduce the cost of erythrocyte preparation, and is beneficial to the development of blood transfusion products. Description of the Drawings

[0013] In order to more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the following will briefly introduce the drawings required for use in the description of the specific embodiments or the prior art. Obviously, the following drawings are some embodiments of the present invention. For those of ordinary skill in the art, other drawings can be obtained based on these drawings without creative efforts.

[0014] Figure 1 It is a schematic flow chart of the method for in vitro induced differentiation of erythrocytes provided in Example 1; Figure 2 It is the proportion result of live cells (FSC, SSC) and erythroid progenitor cells (CD36+ / - GPA-) detected by flow cytometry on the seventh day for each group of differentiation methods in Effect Example 1; Figure 3It is a bar chart of the statistical results of the proportion of erythroid progenitor cells (CD36+ / -GPA-) detected by flow cytometry on the seventh day for each group of differentiation methods in Effect Example 1. ANOVA was used to test for significance. "***" indicates a highly significant difference, where 0.0001 ≤ P < 0.001; Figure 4 It is the results of the proportion of erythroid progenitor cells (CD36+ / - GPA-) detected by flow cytometry on D4 and D7 for each group of differentiation methods in Effect Example 2. T test was used to test for significance. "**" indicates a highly significant difference, where 0.001 ≤ P < 0.01, and "***" indicates a highly significant difference, where 0.0001 ≤ P < 0.001; Figure 5 It is the results of the erythroid colony count on the 10th day of colony culture after taking erythroid progenitor cells from each group of differentiation methods in Effect Example 2 for colony culture on D7. T test was used to test for significance. "*" indicates a significant difference, where 0.01 ≤ P < 0.05; Figure 6 It is the photos of erythroid colonies on the 10th day of colony culture after taking erythroid progenitor cells from each group of differentiation methods in Effect Example 2 for colony culture on D7. The scale of all four photos is 100 μM; Figure 7 It is the total cell number of each group of cells on D0, D7, and D12 in Effect Example 3. ANOVA was used to test for significance. "*" indicates a significant difference, where 0.01 ≤ P < 0.05; Figure 8 It is the proportion of erythrocytes at each stage on the 12th day of differentiation for each group of differentiation methods in Effect Example 3. ANOVA was used to test for significance. "*" indicates a significant difference, where 0.01 ≤ P < 0.05; "**" indicates a highly significant difference, where 0.001 ≤ P < 0.01. Detailed implementation manners

[0015] The technical solutions of the present invention will be clearly and completely described below in conjunction with the embodiments. Obviously, the described embodiments are some, but not all, of the embodiments of the present invention. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0016] In this article, "prepared from..." is synonymous with "comprising". As used herein, the terms "comprising", "including", "having", "containing" or any other variation thereof are intended to cover non-exclusive inclusion. For example, a composition, step, method, article or device containing the listed elements is not necessarily limited to those elements, but may include other elements not explicitly listed or elements inherent to such composition, step, method, article or device.

[0017] In this text, the connecting word "consisting of" excludes any unstated element, step, or component. If used in a claim, this phrase renders the claim closed-ended, excluding materials other than those described, except for conventional impurities associated therewith.

[0018] In this text, when an equivalent, concentration, or other value or parameter is expressed as a range, a preferred range, or a range defined by a series of upper preferred values and lower preferred values, it should be understood that all ranges formed by any pairing of any upper range limit or preferred value with any lower range limit or preferred value are specifically disclosed, regardless of whether the ranges are disclosed individually. For example, when the range "1 to 5" is disclosed, the described range should be interpreted as including the ranges "1 to 4", "1 to 3", "1 to 2", "1 to 2 and 4 to 5", "1 to 3 and 5", etc. When a numerical range is described in this text, unless otherwise stated, the range is intended to include its end values and all integers and fractions within the range.

[0019] In this text, "and / or" is used to indicate that either or both of the stated circumstances may occur. For example, A and / or B includes (A and B) and (A or B).

[0020] In this text, unless otherwise specified, any numbering is used to distinguish one entity or act from another entity or act, rather than necessarily requiring or implying any actual such relationship, order, or degree of importance between these entities or acts. For example, numbers Ⅰ......Ⅴ; first, second... fifth, etc.

[0021] In this text, unless otherwise stated, "optionally", "optional", "optionality", or "option" means that the subsequently described event or circumstance can but does not have to occur, and this description includes the occasions where the event or circumstance occurs or does not occur.

[0022] In this text, "each... independently selected from" can be interchanged with "each... separately and independently selected from" and "each... independently selected from", and all should be understood in a broad sense. It refers to the range or options within which each member of a group of variables or components can be independently selected, that is, the selection of each variable or component is independent and not affected by the selection of other variables or components.

[0023] In this text, a functional derivative refers to a compound obtained by structurally modifying or functionally transforming a parent compound (such as a natural product, bioactive molecule, or synthetic compound) through chemical, physical, or biological methods. For example, by introducing new functional groups, changing the molecular conformation, or binding with other molecules, on the basis of maintaining the core structure and function of the parent compound, a compound with enhanced or expanded partial functions is obtained. Exemplary functional derivatives include, for example, compounds obtained by introducing new functional groups into the parent compound, such as esterification, acylation, or sulfonation; or compounds formed by reacting with acids or bases to form salts, etc.

[0024] In this article, hematopoietic stem and progenitor cells (HSPCs) include hematopoietic stem cells and hematopoietic progenitor cells. Hematopoietic stem cells refer to stem cells that can produce all blood cell types of three hematopoietic lineages (erythroid, lymphoid, and myeloid). Hematopoietic progenitor cells refer to progenitor cells that are formed by the proliferation and differentiation of hematopoietic stem cells under the regulation of a certain microenvironment and certain factors. They are also a type of primitive cells with proliferation ability, but have lost the ability of multi-directional differentiation and can only proliferate and differentiate in a directed manner into one or several blood cell lineages. The said hematopoietic stem cells and hematopoietic progenitor cells express CD34 and CD45. In an alternative embodiment, the hematopoietic stem and progenitor cells include, but are not limited to, pluripotent stem cells, hematopoietic stem and progenitor cells derived from umbilical cord blood, and hematopoietic stem and progenitor cells derived from peripheral blood.

[0025] Erythroid progenitor cells are a group of cells between hematopoietic stem cells and erythroid precursor cells. Erythroid precursor cells are a group of cells between erythroid progenitor cells and mature red blood cells that are differentiated under the action of erythropoietin (EPO). Erythroid progenitor cells and erythroid precursor cells express CD36. Among them, erythroid progenitor cells do not express CD235a, while erythroid precursor cells express CD235a. Red blood cells are enucleated cells with characteristic markers of red blood cell maturation. They specifically express glycoprotein A (CD235a) and do not express the marker CD36.

[0026] The hematopoietic stem and progenitor cells, erythroid progenitor cells, erythroid precursor cells, and reticulocytes in this article can be cells from mammals, including but not limited to humans, monkeys, mice, rats, guinea pigs, rabbits, donkeys, sheep, cows, horses, pigs, or dogs, etc. Human-derived cells are preferred.

[0027] In a first aspect, there is provided the use of a ULK1 inhibitor in any one of (I) to (V): (I) Promoting the expansion of erythroid progenitor cells; (II) Preparing a product for the expansion of erythroid progenitor cells; (III) Inducing the differentiation of red blood cells in vitro; (IV) Preparing a product for inducing the differentiation of red blood cells in vitro; (V) Preparing a blood transfusion product.

[0028] ULK1 (Unc-51-like kinase 1) is a kinase that plays a key role in the process of autophagy. ULK1 inhibitors refer to substances that can inhibit the activity of ULK1. In an alternative embodiment, the ULK1 inhibitor includes SBI-0206965, and the working concentration of SBI-0206965 does not exceed 5 μM, for example, it can be, but is not limited to, not exceeding 4 μM, not exceeding 3 μM, not exceeding 2 μM, or not exceeding 1 μM.

[0029] SBI-0206965 is a potent, selective, and cell-permeable autophagy kinase ULK1 inhibitor that can also inhibit the activity of ULK2 kinase. The molecular formula of SBI-0206965 is C 21 H 21 BrN4O5, CAS number: 1884220-36-3, and the structural formula is shown in Formula (i). In this article, unless otherwise specified, SBI-0206965 also includes its functional derivatives.

[0030] Formula (i).

[0031] In an alternative embodiment, the working concentration of the SBI-0206965 is 0.5 - 1 μM, preferably 1 μM.

[0032] In an alternative embodiment, the (I) includes promoting the expansion of erythroid progenitor cells in the stage of the differentiation of hematopoietic stem and progenitor cells into erythroid progenitor cells.

[0033] In an alternative embodiment, the (II) includes preparing a product for the differentiation of hematopoietic stem and progenitor cells into erythroid progenitor cells.

[0034] In an alternative embodiment, in the (III), it is the in vitro induced differentiation of red blood cells starting from hematopoietic stem and progenitor cells.

[0035] In an alternative embodiment, in the (IV), it is to prepare a product for the in vitro induced differentiation of red blood cells starting from hematopoietic stem and progenitor cells.

[0036] In an alternative embodiment, the application of the first aspect is for non-diagnostic and non-therapeutic purposes.

[0037] Second aspect, a composition for erythrocyte preparation is provided. The composition comprises a first-stage culture composition, and the first-stage culture composition comprises a ULK1 inhibitor, stem cell factor (SCF), erythropoietin (EPO), interleukin-3 (IL-3), holotransferrin, insulin-like growth factor-1 (IGF-1), 3-isobutyl-1-methylxanthine (IBMX), and dexamethasone (DEX). The first-stage culture composition is used to differentiate hematopoietic stem and progenitor cells into erythroid progenitor cells.

[0038] In an optional embodiment, the ULK1 inhibitor comprises SBI-0206965, and the working concentration of SBI-0206965 does not exceed 5 μM, for example, it can be but not limited to not exceeding 4 μM, not exceeding 3 μM, not exceeding 2 μM, or not exceeding 1 μM.

[0039] In an optional embodiment, the working concentration of SCF is 10 - 100 ng / mL, for example, it can be but not limited to 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 ng / mL, preferably 100 ng / mL; and / or, the working concentration of EPO is 1 - 4 U / mL, for example, it can be but not limited to 1, 2, 3, or 4 U / mL, preferably 3 U / mL; and / or, the working concentration of IL-3 is 5 - 10 ng / mL, for example, it can be but not limited to 5, 6, 7, 8, 9, or 10 ng / mL, preferably 10 ng / mL; and / or, the working concentration of holotransferrin is 40 - 60 μg / mL, for example, it can be but not limited to 40, 45, 50, 55, or 60 μg / mL, preferably 50 μg / mL; and / or, the working concentration of IGF-1 is 30 - 50 μg / mL, for example, it can be but not limited to 30, 35, 40, 45, or 50 μg / mL, preferably 40 ng / mL; and / or, the working concentration of IBMX is 40 - 60 μM, for example, it can be but not limited to 40, 45, 50, 55, or 60 μM, preferably 50 μM; and / or, the working concentration of DEX is 0.5 - 1.5 μM, for example, it can be but not limited to 0.5, 0.8, 1, 1.2, or 1.5 μM, preferably 1 μM.

[0040] In an alternative embodiment, the composition further comprises a second-stage culture composition, which comprises SCF, EPO, and holotransferrin. The second-stage culture composition is used to differentiate erythroid progenitor cells into erythroid precursor cells. The second-stage culture composition does not contain a ULK1 inhibitor.

[0041] In an alternative embodiment, the working concentration of SCF is 10-100 ng / mL, such as but not limited to 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 ng / mL, preferably 50 ng / mL; and / or, the working concentration of EPO is 1-4 U / mL, such as but not limited to 1, 2, 3, or 4 U / mL, preferably 3 U / mL; and / or, the working concentration of holotransferrin is 40-60 μg / mL, such as but not limited to 40, 45, 50, 55, or 60 μg / mL, preferably 50 μg / mL.

[0042] In an alternative embodiment, the composition further comprises a third-stage culture composition, which comprises EPO, holotransferrin, human AB serum, insulin, and heparin. The third-stage culture composition is used to differentiate erythroid precursor cells into reticulocytes. The third-stage culture composition does not contain a ULK1 inhibitor.

[0043] In an alternative embodiment, the working concentration of EPO is 1-4 U / mL, such as but not limited to 1, 2, 3, or 4 U / mL, preferably 1 U / mL; and / or, the working concentration of holotransferrin is 0.5-1.5 mg / mL, such as but not limited to 0.5, 1, or 1.5 mg / mL, preferably 1 mg / mL; and / or, the working concentration of human AB serum is 1-10% v / v, such as but not limited to 1, 2, 5, 8, or 10% v / v, preferably 5% v / v; and / or, the working concentration of insulin is 1-10 μg / ml, such as but not limited to 1, 2, 5, 8, or 10 μg / ml, preferably 5 μg / ml; and / or, the working concentration of heparin is 1-5 U / mL, such as but not limited to 1, 2, 3, 4, or 5 U / mL, preferably 3 U / mL.

[0044] In an alternative embodiment, the first-stage culture composition, the second-stage culture composition, and the third-stage culture composition each independently further comprise an antibiotic, such as but not limited to penicillin and streptomycin.

[0045] In a third aspect, a culture medium for erythrocyte preparation is provided, which comprises the composition described in the second aspect and a basal medium. The basal medium refers to a culture medium that provides general nutrients and a culture environment for cells, and the specific type of the basal medium is not limited in the present invention.

[0046] In an optional embodiment, the culture medium for erythrocyte preparation comprises a first-stage culture medium, a second-stage culture medium, and a third-stage culture medium. The first-stage culture medium comprises the first-stage culture composition described in the second aspect and a basal medium; the second-stage culture medium comprises the second-stage culture composition described in the second aspect and a basal medium; the third-stage culture medium comprises the third-stage culture composition described in the second aspect and a basal medium. The basal media in each stage of the culture medium can be the same or different.

[0047] In an optional embodiment, the first-stage culture medium comprises 0.5 - 1 μM SBI-0206965, 10 - 100 ng / mL SCF, 1 - 4 U / mL EPO, 5 - 10 ng / mL IL-3, 40 - 60 μg / mL holo-transferrin, 30 - 50 ng / mL IGF-1, 40 - 60 μM IBMX, and 0.5 - 1.5 μM Dex.

[0048] In an optional embodiment, the second-stage culture medium comprises 50 ng / ml SCF, 3 U / ml EPO, and 50 μg / ml holo-transferrin.

[0049] In an optional embodiment, the third-stage culture medium comprises 1 U / ml EPO, 1 mg / ml holo-transferrin, 5% human AB serum, 5 μg / ml insulin, and 3 U / m heparin.

[0050] In an optional embodiment, the first-stage culture medium, the second-stage culture medium, and the third-stage culture medium each independently further contain 100 U / mL penicillin and 100 μg / mL streptomycin.

[0051] In a fourth aspect, a method for differentiating hematopoietic progenitor cells into erythroid progenitor cells is provided, the method comprising culturing hematopoietic progenitor cells using a differentiation medium containing a ULK1 inhibitor to differentiate the hematopoietic progenitor cells into erythroid progenitor cells.

[0052] In an optional embodiment, the ULK1 inhibitor comprises SBI-0206965, and the working concentration of SBI-0206965 does not exceed 5 μM, for example, it can be but not limited to not exceeding 4 μM, not exceeding 3 μM, not exceeding 2 μM, or not exceeding 1 μM.

[0053] In an alternative embodiment, the working concentration of SBI-0206965 is 0.5 to 1 μM, preferably 1 μM.

[0054] In an alternative embodiment, hematopoietic stem and progenitor cells are cultured in a medium containing the first-stage culture composition described in the second aspect to differentiate the hematopoietic stem and progenitor cells into erythroid progenitor cells.

[0055] In a fifth aspect, a method for in vitro induction of differentiation of erythrocytes is provided. The method includes using the erythroid progenitor cells obtained by the method described in the fourth aspect, and then culturing the erythroid progenitor cells in a differentiation induction medium without a ULK1 inhibitor to differentiate the erythroid progenitor cells into erythroid precursor cells. The differentiation induction medium can be selected from the media known and conventional in the art for the continuous differentiation of erythroid progenitor cells into erythrocytes, and the present invention places no limitation thereon.

[0056] In an alternative embodiment, the method for in vitro induction of differentiation of erythrocytes includes in vitro induction of differentiation of erythrocytes using the medium described in the fourth aspect, including culturing hematopoietic stem and progenitor cells in a medium containing the first-stage culture composition to differentiate the hematopoietic stem and progenitor cells into erythroid progenitor cells; culturing the erythroid progenitor cells in a medium containing the second-stage culture composition to differentiate the erythroid progenitor cells into erythroid precursor cells; and culturing the erythroid precursor cells in a medium containing the third-stage culture composition to differentiate the erythroid precursor cells into reticulocytes.

[0057] In an alternative embodiment, SBI-0206965 in any of the first, second, third, fourth, and fifth aspects is SBI-0206965 as shown in formula (i), and the CAS number is 1884220-36-3.

[0058] The present invention will be further illustrated by specific examples below. However, it should be understood that these examples are only for more detailed illustration and should not be construed as limiting the present invention in any form.

[0059] Example 1 This example provides a method for in vitro induction of differentiation of erythrocytes. The process schematic diagram is as Figure 1 shown and includes the following steps: (1) In the first stage of differentiation, hematopoietic stem and progenitor cells (CD34 5 cells) derived from umbilical cord blood are seeded into the erythroid differentiation medium of the first stage at a density of 1×10 + cells / mL and differentiated for 7 days to obtain a mixed culture of erythroid progenitor cells and erythroid precursor cells.

[0060] The components of the erythrocyte differentiation medium in the first stage are: 0.5 μM SBI-0206965, 100 ng / mL stem cell factor (SCF), 3 U / mL erythropoietin (EPO), 10 ng / mL interleukin-3 (IL-3), 50 μg / mL holotransferrin, 40 ng / mL insulin-like growth factor-1 (IGF-1), 50 μM 1-methyl-3-isobutylxanthine (IBMX), 1 μM dexamethasone (DEX), 100 U / mL penicillin, 100 μg / mL streptomycin. Fluid replacement is carried out every two days, and medium replacement is carried out every 3 or 4 days.

[0061] (2) In the second stage of differentiation, the culture product of step (1) is seeded into the erythroid differentiation medium in the second stage at a density of 1×10 5 cells / mL. Fluid replacement is carried out every two days, and medium replacement is carried out every 3 or 4 days. After 5 days of differentiation, more erythroid progenitor cells are obtained.

[0062] The components of the erythrocyte differentiation medium in the second stage are: 50 ng / mL SCF, 3 U / mL EPO, 50 μg / mL holotransferrin, 100 U / mL penicillin, 100 μg / mL streptomycin.

[0063] (3) In the third stage of differentiation, the culture product of step (2) is seeded into the erythroid differentiation medium in the third stage at a density of 0.5 - 1×10 6 cells / mL. Fluid replacement is carried out every two days, and medium replacement is carried out every 3 or 4 days. After 9 days of differentiation.

[0064] The components of the erythroid differentiation medium in the third stage are: 1 U / mL EPO, 1 mg / mL holotransferrin, 5% human AB serum, 5 μg / mL insulin, 3 U / mL heparin, 100 U / mL penicillin, 100 μg / mL streptomycin.

[0065] Example 2 This example provides a method for in vitro induction of erythrocyte differentiation. The difference from Example 1 is that 1 μM SBI-0206965 is added to the erythrocyte differentiation medium in the first stage.

[0066] Example 3 This example provides a method for in vitro induced differentiation of red blood cells. The difference from Example 2 is only in the first stage of differentiation. Hematopoietic stem and progenitor cells (CD34 4 cells) are seeded into the erythroid differentiation medium in the first stage at a density of 1×10 + cells / mL and differentiated for 7 days.

[0067] Example 4 This example provides a method for in vitro induced differentiation of red blood cells. The difference from Example 1 is only that the components of the erythroid differentiation medium in the first stage are: 10 ng / mL SCF, 4 U / mL EPO, 5 ng / mL IL-3, 50 μg / mL holotransferrin, 40 ng / mL IGF-1, 50 μM IBMX, 1 μM DEX, 100 U / mL penicillin, 100 μg / mL streptomycin, and 1 μM SBI-0206965 is added during this process.

[0068] Example 5 This example provides a method for in vitro induced differentiation of red blood cells. The difference from Example 1 is only that the components of the erythroid differentiation medium in the first stage are: 100 ng / mL SCF, 1 U / mL EPO, 10 ng / mL IL-3, 50 μg / mL holotransferrin, 40 ng / mL IGF-1, 50 μM IBMX, 1 μM DEX, 100 U / mL penicillin, 100 μg / mL streptomycin, and 1 μM SBI-0206965 is added during this process.

[0069] Comparative Example 1 This comparative example provides a method for in vitro induced differentiation of red blood cells. The difference from Example 1 is only that 5 μM SBI-0206965 is added to the erythroid differentiation medium in the first stage.

[0070] Comparative Example 2 This comparative example provides a method for in vitro induced differentiation of red blood cells. The difference from Example 1 is only that 10 μM SBI-0206965 is added to the erythroid differentiation medium in the first stage.

[0071] Comparative Example 3 This comparative example provides a method for in vitro induced differentiation of red blood cells. The difference from Example 1 is only that SBI-0206965 is not added to the erythroid differentiation medium in the first stage.

[0072] Comparative Example 4 This comparative example provides a method for in vitro induction and differentiation of red blood cells. The difference from Example 3 is only that 1 μM SBI-0206965 is added to the red blood cell differentiation medium in the second stage.

[0073] Comparative Example 5 This comparative example provides a method for in vitro induction and differentiation of red blood cells. The difference from Example 3 is only that SBI-0206965 is not added to the red blood cell differentiation medium in the first stage.

[0074] Effect Example 1 Different concentrations of SBI-0206965 (0 μM, 0.5 μM, 1 μM, 5 μM, and 10 μM, corresponding to Comparative Example 3, Example 1, 2, and Comparative Examples 1, 2) were added in the first stage of differentiation. On the 7th day (D7), flow cytometry was used to detect the proportions of live cells (FSC, SSC) and erythroid progenitor cells (CD36+ / - GPA-). The detection found that in the range of 0.5 - 1 μM addition, without affecting the proportion of live cells ( Figure 2 in a), the proportion of erythroid progenitor cells ( Figure 2 in b and Figure 3 ), increased. While at concentrations of 5 - 10 μM, the proportions of both live cells and erythroid progenitor cells decreased ( Figure 2 in b and Figure 3 ). Therefore, adding 0.5 - 1 μM SBI-0206965 to the red blood cell differentiation medium in the first stage can promote the differentiation of hematopoietic stem and progenitor cells into erythroid progenitor cells without affecting cell viability, and the effect is best when adding 1 μM SBI-0206965.

[0075] Effect Example 2 Taking Comparative Example 3 as the control group, flow cytometry was used to detect the proportion of erythroid progenitor cells (CD36+ / -GPA-) in the induction and differentiation methods of Example 2 and Comparative Example 3 on the 4th day (D4) and the 7th day (D7) of the first differentiation stage. The results are as Figure 4 shown. And on the 7th day (D7) of the differentiation methods of Example 2 and Comparative Example 3, 200 cells / dish were taken for colony culture (Stemcell, H4435). On the 10th day of colony culture, the erythroid colonies were counted and photographed. The counting results are as Figure 5 shown, and the photographs of erythroid colonies are as Figure 6 shown, Figure 6 and the left and right column photos in Figure 4 are the experimental results of two repeated experiments. The results show that adding 1 μM SBI-0206965 in the first stage of differentiation can generate more erythroid progenitor cells ( Figure 5 ), and the ability of erythroid progenitor cells to produce red blood cells is enhanced ( Figure 6 ).

[0076] Effect Example 3 With 1×10 4 CD34+ cells per group, divided into three groups. One group was the control group (Comparative Example 5), one group added only 1 μM SBI-0206965 in the first stage (Example 3), and one group added 1 μM SBI-0206965 in the first and second stages (Comparative Example 4). The total cell number was counted on day 0 (D0), day 7 (D7), and day 12 (D12) of differentiation ( Figure 7 ), and the proportion of red blood cells at each stage on day 12 of differentiation was detected by flow cytometry ( Figure 8 ).

[0077] The results showed that in Example 3, SBI-0206965 was added only in the first differentiation stage, and the drug was withdrawn and cultured until D12 in the second differentiation stage. The total number of red blood cells increased significantly, and there was no significant difference in the proportion of red blood cells at subsequent stages. If SBI-0206965 was continuously added in the first and second differentiation stages (Comparative Example 4), there was no significant difference in the total number of red blood cells compared with the control group ( Figure 7 ), and the proportions of late proerythroblasts, basophilic erythroblasts, and orthochromatic erythroblasts decreased significantly ( Figure 8 ). Therefore, SBI-0206965 can only be added in the first differentiation stage.

[0078] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that they can still modify the technical solutions described in the foregoing embodiments, or perform equivalent replacements on some or all of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. Use of ULK1 inhibitor in any one of (I) to (V): (I) Promoting the expansion of erythroid progenitor cells; (II) Preparing a product for promoting the expansion of erythroid progenitor cells; (III) Inducing the differentiation of erythrocytes in vitro; (IV) Preparing a product for inducing the differentiation of erythrocytes in vitro; (V) Preparing a blood transfusion product.

2. The application according to claim 1, wherein The ULK1 inhibitor includes SBI-0206965, and the working concentration of SBI-0206965 does not exceed 5 μM.

3. A composition for red blood cell preparation, characterized in that, Comprising a first-stage culture composition, the first-stage culture composition comprising a ULK1 inhibitor, stem cell factor, erythropoietin, interleukin-3, holotransferrin, insulin-like growth factor-1, 3-isobutyl-1-methylxanthine, and dexamethasone.

4. The composition according to claim 3, wherein The ULK1 inhibitor includes SBI-0206965, and the working concentration of SBI-0206965 does not exceed 5 μM.

5. The composition according to claim 3 or 4, characterized in that, Further comprising a second-stage culture composition, and / or a third-stage culture composition; The second-stage culture composition comprises stem cell factor, erythropoietin, and holotransferrin; The third-stage culture composition comprises erythropoietin, holotransferrin, human AB serum, insulin, and heparin; The second-stage culture composition and the third-stage culture composition do not contain a ULK1 inhibitor.

6. Culture medium for red blood cell preparation, characterized in that, Comprising the composition according to any one of claims 3 to 5 and a basal medium.

7. A method for differentiating hematopoietic stem and progenitor cells into erythroid progenitor cells, characterized in that, Including culturing hematopoietic stem progenitor cells with a differentiation medium containing a ULK1 inhibitor to differentiate the hematopoietic stem progenitor cells into erythroid progenitor cells.

8. The method according to claim 7, characterized in that, The ULK1 inhibitor includes SBI-0206965, and the working concentration of SBI-0206965 does not exceed 5 μM; and / or, Culturing hematopoietic stem progenitor cells with a medium containing the first-stage culture composition according to claim 3 or 4 to differentiate the hematopoietic stem progenitor cells into erythroid progenitor cells.

9. A method for in vitro induced differentiation of red blood cells, characterized in that, Including obtaining erythroid progenitor cells by the method according to claim 7 or 8, and then culturing the erythroid progenitor cells in a differentiation induction medium without a ULK1 inhibitor.

10. The method for in vitro induced differentiation of red blood cells according to claim 9, wherein, Including inducing the differentiation of erythrocytes in vitro using the medium according to claim 6; Culturing hematopoietic stem progenitor cells with a medium containing the first-stage culture composition to differentiate the hematopoietic stem progenitor cells into erythroid progenitor cells; culturing erythroid progenitor cells with a medium containing the second-stage culture composition to differentiate the erythroid progenitor cells into erythroid precursor cells; culturing erythroid precursor cells with a medium containing the third-stage culture composition to differentiate the erythroid precursor cells into reticulocytes.

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