CN116622631A - Application of RO8191 and/or AS2863619 in promoting differentiation and proliferation of erythroid progenitor cells or erythroid precursor cells - Google Patents

Application of RO8191 and/or AS2863619 in promoting differentiation and proliferation of erythroid progenitor cells or erythroid precursor cells Download PDF

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CN116622631A
CN116622631A CN202310907811.9A CN202310907811A CN116622631A CN 116622631 A CN116622631 A CN 116622631A CN 202310907811 A CN202310907811 A CN 202310907811A CN 116622631 A CN116622631 A CN 116622631A
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cells
erythroid
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progenitor cells
erythrocyte
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CN116622631B (en
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杜如龙
于蕾
武雪宁
黄雯静
张成志
郜华磊
吴理达
顾雨春
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Chengnuo Regenerative Medical Technology Beijing Co ltd
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Abstract

The invention discloses application of RO8191 and/or AS2863619 in promoting differentiation and proliferation of erythroid progenitor cells or erythroid precursor cells, and discovers that RO8191 and AS2863619 can promote differentiation, proliferation and long-time maintenance of erythroid progenitor cells or erythroid precursor cells for the first time, and the combination of RO8191 and AS2863619 has a synergistic effect, solves the problems that erythroid progenitor cells and erythroid precursor cells are low in generation efficiency and erythroid progenitor cells and erythroid precursor cells cannot be effectively amplified in the erythroid progenitor cells induced differentiation process in the prior art, and has good application prospect.

Description

Application of RO8191 and/or AS2863619 in promoting differentiation and proliferation of erythroid progenitor cells or erythroid precursor cells
Technical Field
The invention belongs to the technical field of biological medicines, and particularly relates to application of RO8191 and/or AS2863619 in promoting erythroid progenitor cells or erythroid precursor cells to differentiate and proliferate.
Background
Maintaining a constant oxygen supply to tissues is critical to the survival of many organisms, particularly humans. Red Blood Cells (RBCs) transport oxygen in the body through the Blood stream, which is provided by hemoglobin, a protein specific to Red Blood cells that is capable of binding oxygen. When the red blood cells reach the tissue, oxygen diffuses through the capillary walls. Therefore, the effect of erythrocytes is of paramount importance. Erythrocyte infusion is currently an important clinical treatment and effective means for treating severe anemia and acute blood loss, and so far, erythrocyte infusion is still mainly based on blood from the donor. The current studies indicate that mature erythrocytes can be obtained by in vitro induction of human isolated cd34+ hematopoietic stem cells (Hemopoietic Stem Cell, HSCs). However, the hematopoietic stem cells have a low in vivo proportion and limited in vitro expansion capacity, so that a large number of mature red blood cells cannot be obtained, and the requirement of clinical blood cannot be fundamentally met.
With the rapid development of stem cell research and regenerative medicine, new cell therapies derived and developed based on stem cells have become the most promising applications for solving the major clinical diseases faced by humans. Human pluripotent stem cells (Human Pluripotent Stem Cell, hpscs) are a class of cells with self-renewing and differentiating potential that can achieve in vitro unlimited proliferation and directed differentiation of all human cells. Therefore, the directional induction and differentiation of human pluripotent stem cells into mature erythrocytes as a clinical blood substitute in vitro is possible to provide a new scheme and thought for solving the current clinical transfusion shortage and safety problems. However, there are still many critical technical problems to be solved in vitro to induce mature erythrocytes and finally apply them to clinic, including low efficiency of directional induction differentiation of hematopoietic stem cells into erythrocytes, low efficiency of erythrocyte maturation and enucleation, difficulty in mass production of mature erythrocytes, etc., and efficiency of erythroid progenitor cells or erythroid precursor cells proliferation is an important factor affecting the efficiency of directional differentiation, maturation and enucleation of erythrocytes.
The low induction of erythroid progenitor cells or erythroid precursor cells is not favorable for mass production of erythrocytes, and cannot fundamentally meet the demand of clinical blood. Therefore, how to solve the generation and long-time expression of erythroid progenitor cells or erythroid precursor cells in the erythroid induced differentiation process becomes one of the difficulties in the field of erythroid in vitro induced differentiation. At present, no related study or report on the promotion of erythrocyte differentiation of RO8191 and/or AS2863619 is known.
Disclosure of Invention
In view of this, in order to overcome the above-mentioned technical problem of low proliferation efficiency of erythroid progenitor cells or erythroid precursor cells in the prior art, the present invention aims to provide an application of RO8191 and/or AS2863619 in promoting differentiation and proliferation of erythroid progenitor cells or erythroid precursor cells.
In order to achieve the above purpose, the present invention adopts the following technical scheme:
in a first aspect the invention provides the use of RO8191 and/or AS2863619 for promoting the differentiation and proliferation of erythroid progenitor cells or erythroid precursor cells.
In the invention, RO8191 is an imidazonaphthyridine compound, the structural formula is shown in formula (I), and the name is: 8- (1, 3, 4-oxazol-2-yl) -2,4-bis (trifluoromethyl) imidazo [1,2-a ] [1,8] naphthyridine, CAS number: 691868-88-9, molecular formula: C14H5F6N5O is an orally active, potent Interferon (IFN) receptor agonist that binds directly to ifnα/β receptor 2, activating IFN-stimulated gene (ISGs) expression and JAK/STAT phosphorylation. RO8191 shows antiviral activity against both HCV and EMCV with an IC50 of 200 nM for HCV replicon. RO8191 acts as a cccDNA modulator by having interferon-like activity and has anti-HBV activity.
Formula (I).
In the present invention, the AS2863619 is a potent, oral cyclin-dependent kinase 8 (CDK 8) and CDK19 inhibitor, and has the structural formula shown in formula (II), and is named: 4- (1- (2-Methyl-1H-benzol [ d ] imidazol-6-yl) -1H-imidazol [4,5-c ] pyridin-2-yl) -1,2, 5-oxazo l-3-amine dihydrochloride, CAS number: 2241300-51-4, molecular formula: C16H14Cl2N8O, AS2863619 can convert antigen specific effectors or memory T cells into foxp3+ regulatory T (Treg) cells to study various immune diseases with IC50 for CDK8 inhibitors and CDK19 inhibitors of 0.61 nM and 4.28 nM, respectively. Inhibition of CDK8/19 by AS2863619 enhances STAT5 activation, thereby activating the Foxp3 gene.
Formula (II).
In some embodiments, the invention verifies through experiments that RO8191 and/or AS2863619 can promote differentiation, proliferation and long-time maintenance of erythroid progenitor cells or erythroid precursor cells for the first time, wherein the combination of RO8191 and AS2863619 has a synergistic effect on the differentiation, proliferation and long-time maintenance of erythroid progenitor cells or erythroid precursor cells, and can significantly improve the induced differentiation efficiency of erythroid progenitor cells and erythroid precursor cells in the induced differentiation process of erythrocytes.
In a second aspect, the invention provides a basal medium for promoting the induced differentiation of hematopoietic stem cells or hematopoietic progenitor cells into erythroid progenitor cells or erythroid precursor cells.
Further, the basal medium comprises RO8191 and/or AS2863619.
Further, the basal medium also comprises IMDM medium, ITS-X, ascorbic acid, acetylcysteine, quinine dimethacrylate and BSA.
Further, the contents of each component in the basic culture medium are respectively as follows: 5. mu M RO8191, 1 mu M AS2863619, 1% ITS-X, 50 mu g/mL ascorbic acid, 50 mu M acetylcysteine, 50 mu M quinio dimethacrylate, 0.5% BSA.
In some embodiments, the basal medium comprises the following components: (0.5-20) [ mu ] M RO8191, (0.1-10) [ mu ] M AS2863619, (0.1-10)% ITS-X, (10-100) [ mu ] g/mL ascorbic acid, (10-100) [ mu ] M acetylcysteine, (10-100) [ mu ] M quinioDi-methacrylate, (0.1-10)% BSA.
In a specific embodiment, the content of each component in the basal medium is respectively as follows: 5. mu M RO8191, 1 mu M AS2863619, 1% ITS-X, 50 mu g/mL ascorbic acid, 50 mu M acetylcysteine, 50 mu M quinio dimethacrylate, 0.5% BSA.
In some embodiments, the hematopoietic stem cells or hematopoietic progenitor cells include hematopoietic stem cells or hematopoietic progenitor cells of various origins, including, but not limited to: cord blood-derived hematopoietic stem/progenitor cells (CB-HSPC), induced pluripotent stem/progenitor cells (hiPSC-HSPC), bone marrow-derived hematopoietic stem/progenitor cells, peripheral blood-derived hematopoietic stem/progenitor cells. Hematopoietic stem cells or hematopoietic progenitor cells of various origins are within the scope of the invention.
In the present invention, the erythroid progenitor cells (Erythroid progenitor cells) are a group of cells between hematopoietic stem cells and erythroid precursor cells, and the stage of differentiation from erythroid progenitor cells to erythroid precursor cells is a key process in regulating the erythropoiesis homeostasis mechanism. The erythroid precursor cells (Erythroid precursor cells) are a cell population between erythroid progenitor cells and erythrocytes, which is differentiated under the action of Erythropoietin (EPO). The erythroid progenitor cells and erythroid precursor cells express CD36, wherein the erythroid progenitor cells do not express CD235a and the erythroid precursor cells express CD235a.
In the present invention, the erythrocytes are enucleated cells with a marker characteristic of erythrocyte maturation, which express in particular glycoprotein a (CD 235 a), without expressing the marker CD36. Red blood cells are the most abundant type of blood cells in the blood and are the most prominent vehicle 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, accounting for 90% of erythrocytes. Hemoglobin is a heme-containing protein molecule that binds oxygen molecules in the lungs or gills and then releases the bound oxygen molecules in the body's tissues.
In the present invention, the hematopoietic stem cells refer to stem cells capable of producing all blood cell types of three hematopoietic lineages (erythroid, lymphoid and myeloid), including myeloid lineages (monocytes and macrophages, neutrophils, basophils, eosinophils, erythrocytes, megakaryocytes/platelets, dendritic cells) and lymphoid lineages (T cells, B cells, NK cells). The hematopoietic progenitor cells refer to progenitor cells which proliferate and differentiate into various blood cells under the regulation of a certain microenvironment and certain factors, are also quite primitive cells with proliferation capability, but have lost the multi-directional differentiation capability, can only proliferate and differentiate to one or several blood cell lines in a targeted way, and can differentiate into multi-functional cells of several cell types of the hematopoietic system, including but not limited to: granulocytes, monocytes, erythrocytes, megakaryocytes, B cells and T cell hematopoietic progenitors are committed to the hematopoietic lineage and generally do not self-regenerate. The hematopoietic stem cells and hematopoietic progenitor cells express CD45.
In a third aspect, the invention provides a culture system for promoting the induced differentiation of hematopoietic stem cells or hematopoietic progenitor cells into erythroid progenitor cells or erythroid precursor cells.
Further, the culture system comprises a erythrocyte specialization stage culture medium, a erythrocyte expansion stage culture medium and a erythrocyte maturation stage culture medium;
the erythrocyte characterization stage medium comprises the basic medium, SCF, IL-3, EPO, dexamethasone according to the second aspect of the invention;
the erythrocyte expansion stage culture medium comprises the basic culture medium, SCF, IL-3, EPO and dexamethasone according to the second aspect of the invention;
the erythrocyte maturation stage culture medium comprises the basal culture medium, SCF and EPO according to the second aspect of the invention.
Further, the content of each component in the erythrocyte-specific stage culture medium is as follows: 50 ng/mL SCF, 10 ng/mL IL-3, 10 ng/mL EPO, 1 [ mu ] M dexamethasone;
the erythrocyte expansion stage culture medium comprises the following components in percentage by weight: 50 ng/mL SCF, 10 ng/mL IL-3, 10 ng/mL EPO, 1 [ mu ] M dexamethasone;
the content of each component in the culture medium at the erythrocyte maturation stage is respectively as follows: 50 ng/mL SCF, 10 ng/mL EPO.
In some embodiments, the red blood cell specification stage medium comprises the following components: (10-100) ng/mL SCF, (1-50) ng/mL IL-3, (1-50) ng/mL EPO, (0.1-10) [ mu ] M dexamethasone.
In a specific embodiment, the content of each component in the erythrocyte-specific stage medium is respectively as follows: 50 ng/mL SCF, 10 ng/mL IL-3, 10 ng/mL EPO, 1 [ mu ] M dexamethasone.
In some embodiments, the red blood cell expansion stage medium comprises the following components: (10-100) ng/mL SCF, (1-50) ng/mL IL-3, (1-50) ng/mL EPO, (0.1-10) [ mu ] M dexamethasone.
In a specific embodiment, the erythrocyte expansion stage medium comprises the following components in percentage by weight: 50 ng/mL SCF, 10 ng/mL IL-3, 10 ng/mL EPO, 1 [ mu ] M dexamethasone.
In some embodiments, the red blood cell maturation stage medium comprises the following components: (10-100 ng/mL SCF, (1-50 ng/mL EPO).
In a specific embodiment, the content of each component in the erythrocyte maturation stage culture medium is respectively as follows: 50 ng/mL SCF, 10 ng/mL EPO.
In some embodiments, the numerical ranges or specific values recited herein (e.g., the content ranges or specific amounts of the components in the basal medium, the content ranges or specific amounts of the various induced differentiation factors added in the respective differentiation stages, the cell density ranges or specific values in the respective differentiation stages, the number of days of culture in the respective differentiation stages, the number of medium exchanges, etc.) are merely for explaining the technical effects achieved by the present invention, and are not to be construed as limitations of the present invention, and those of ordinary skill in the art may make various adjustments or modifications on the basis of the numerical ranges or specific values recited herein, so long as the intended induced differentiation effects can be achieved, and such adjusted or modified data ranges or specific values are also included in the scope of the present invention.
In a fourth aspect, the invention provides a method of promoting the induced differentiation of hematopoietic stem cells or hematopoietic progenitor cells into erythroid progenitor cells or erythroid precursor cells.
Further, the method comprises culturing the hematopoietic stem cells or hematopoietic progenitor cells using a culture system according to the third aspect of the invention.
Further, the method comprises the following steps:
(1) Day0-6, a erythrocyte-specific stage, culturing hematopoietic stem cells or hematopoietic progenitor cells using the erythrocyte-specific stage medium of the third aspect of the invention;
(2) Day6-12, culturing the cells obtained in step (1) using the erythrocyte expansion phase medium of the third aspect of the present invention;
(3) Day12-15, erythroid progenitor cells or erythroid precursor cells are obtained by culturing the cells obtained in step (2) in the erythroid maturation stage medium according to the third aspect of the present invention.
Further, the medium used during Day0-3, day0-6, day0-9, day0-12 or Day0-15 contains RO8191 and AS2863619.
Further, the cell density in step (1) was 1X 105 cells/mL;
the cell density in step (2) was 5X 105 cells/mL;
the cell density in step (3) was 1X 106 cells/mL.
Further, fresh medium was changed every 2 days until Day20.
In some embodiments, the present invention has been experimentally verified to be able to significantly increase the differentiation efficiency of CD36+CD235a+ erythroid precursor cells by adding RO8191 and AS2863619 to the medium used during Day0-3, day0-6, day0-9, day0-12 or Day0-15, for the first time, and the earlier and longer duration of action, the higher the differentiation efficiency of CD36+CD23235a+ erythroid precursor cells, and in addition, the longer duration of action, the higher the differentiation efficiency of CD36+CD2323235a-erythroid precursor cells, i.e., the synergistic effect of RO8191 and AS2863619 at an early and long-term induced differentiation, is more advantageous for increasing the induced differentiation of erythroid progenitor cells and erythroid precursor cells.
In some embodiments, the cell density in step (1) is (0.01-10). Times.105 cells/mL, and in particular embodiments, the cell density in step (1) is 1X 105 cells/mL.
In some embodiments, the cell density in step (2) is (0.1-50). Times.105 cells/mL, and in particular embodiments, the cell density in step (2) is 5X 105 cells/mL.
In some embodiments, the cell density in step (3) and step (4) is (0.01-10). Times.106 cells/mL, and in particular embodiments, the cell density in step (3) and step (4) is 1X 106 cells/mL.
In addition, the invention also provides a method for inducing hematopoietic stem cells or hematopoietic progenitor cells to differentiate into erythrocytes.
Further, the method comprises the following steps:
(1) Day0-6, a erythrocyte-specific stage, culturing hematopoietic stem cells or hematopoietic progenitor cells using the erythrocyte-specific stage medium of the third aspect of the invention;
(2) Day6-12, culturing the cells obtained in step (1) using the erythrocyte expansion phase medium of the third aspect of the present invention;
(3) Day12-15, culturing the cells obtained in step (2) using the erythrocyte maturation stage medium of the third aspect of the present invention;
(4) Day15-20, culturing the cells obtained in the step (3) by adopting a medium in the erythrocyte enucleation stage to obtain the erythrocytes.
Further, the erythrocyte enucleation stage medium in step (4) comprises IMDM medium, ITS-X, ascorbic acid, acetylcysteine, quinine dimethacrylate, BSA, EPO.
In some embodiments, the red blood cell enucleation stage medium comprises the following components: (0.1-10)% ITS-X, (10-100) mug/mL ascorbic acid, (10-100) mug acetylcysteine, (10-100) mug quinio dimethacrylate, (1-50) ng/mL EPO, (0.1-10)% BSA,
In a specific embodiment, the content of each component in the erythrocyte enucleation stage culture medium is as follows: 1% ITS-X, 50 [ mu ] g/mL ascorbic acid, 50 [ mu ] M acetylcysteine, 50 [ mu ] M quinio dimethacrylate, 0.5% BSA, 10 ng/mL EPO.
Further, the medium used during Day0-3, day0-6, day0-9, day0-12 or Day0-15 contains RO8191 and AS2863619.
In addition, the invention also provides the application of the red blood cells obtained by inducing differentiation based on the method for inducing the differentiation of the hematopoietic stem cells or hematopoietic progenitor cells into the red blood cells in preventing, treating and/or improving the red blood cell related diseases or disorders.
In addition, the present invention provides a method for preventing, treating and/or ameliorating a red blood cell-related disease or disorder, the method comprising administering to a subject in need thereof red blood cells induced to differentiate based on the method for inducing differentiation of hematopoietic stem cells or hematopoietic progenitor cells into red blood cells as described above.
Further, the red blood cell related diseases or disorders refer to those diseases or disorders requiring the infusion of red blood cells, including but not limited to: severe anemia, acute blood loss, cancer, transplantation, autoimmune diseases, infectious diseases, inflammation, immunodeficiency related diseases, and the like.
In some embodiments, the erythrocytes are administered to the subject by one or more routes selected from systemic, local, intravenous, subcutaneous, intra-articular, intramuscular, intrathecal, and intraperitoneal. In some embodiments, the subject comprises one or more animals, including, for example, bovine, equine, ovine, primate, avian, and rodent species. The subject may be an animal (e.g., mammal, bird, fish, reptile, or amphibian) that includes red blood cells in its blood. In some embodiments, the subject may be a mammal, such as a human or non-human mammal. In other embodiments, the subject may 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 a human.
Compared with the prior art, the invention has the advantages and beneficial effects that:
the invention discovers that RO8191 and AS2863619 can promote differentiation, proliferation and long-time maintenance of CD36+ erythroid progenitor cells or erythroid precursor cells for the first time, and the combination of RO8191 and AS2863619 has a synergistic effect, solves the problems that the erythroid progenitor cells and erythroid precursor cells are low in generation efficiency and cannot effectively realize the expansion of erythroid progenitor cells and erythroid precursor cells in the erythroid induced differentiation process in the prior art, and the research discovers that the research of inducing differentiation of hematopoietic stem progenitor cells from different sources into erythrocytes is greatly promoted, the mass production and preparation of in-vitro erythrocytes are promoted, and the problems of clinical blood shortage and safety are finally solved.
Drawings
FIG. 1 is a graph showing the effect of Garcinone D, RO8191 and AS2863619 on erythrocyte induced differentiation, wherein, in the graph A: cell flow analysis results of effects of 5 μm Garcinone D, 5 μm RO8191 and 1 μm AS2863619 on erythroid progenitor markers CD36, CD71 and erythroid marker CD235a, B panels: 5. results of influence of [ mu ] M Garcinone D, 5 [ mu ] M RO8191 and 1 [ mu ] M AS2863619 on cell morphology change are graphs, and Control is a blank Control group; gd+as+ro is an experimental group representing the addition of 5 μm Garcinone D, 5 μm RO8191 and 1 μm AS2863619 during Day 0-12;
FIG. 2 is a graph showing the effect of Garcinone D, RO8191 and AS2863619 on erythrocyte induced differentiation, wherein, in the graph A: results of induced differentiation day9 cell flow analysis 5 μm Garcinone D, 5 μm RO8191 or/and 1 μm AS2863619 effect on hematopoietic stem progenitor markers CD34, CD45, erythroid progenitor markers CD36, CD71 and erythroid marker CD235a, panel B: on the 15 th day of induced differentiation, 5 mu M Garcinone D, 5 mu M RO8191 or/and 1 mu M AS2863619 influence result graphs of hematopoietic stem progenitor cell markers CD34 and CD45, erythroid progenitor cell markers CD36 and CD71 and erythrocyte marker CD235a, wherein Control is a blank Control group; GD stands for adding 5 μm Garcinone D during Day 0-12; RO stands for adding 5 mu M RO8191 during Day 0-12; AS stands for 1 mu M AS2863619 added during Day 0-12; RO+AS represents the simultaneous addition of 5 [ mu ] M RO8191 and 1 [ mu ] M AS2863619 during Day 0-12;
FIG. 3 is a graph showing the results of a cell flow assay of the effect of 1. Mu.M AS2863619 or/and 5. Mu.M RO8191 on hematopoietic stem progenitor markers CD34, CD45, erythroid progenitor markers CD36, CD71, and erythroid marker CD235a, wherein Control is a blank; AS stands for Day0-12 handling 1 [ mu ] M AS2863619; RO+AS represents the treatment with 5 [ mu ] M RO8191 on the basis of the treatment with 1 [ mu ] M AS2863619 of Day0-3, day0-6, day0-9, day0-12, respectively;
fig. 4 is a graph of the results of a time window search for RO8191 in conjunction with AS2863619 to promote erythroid progenitor and erythroid precursor cell production, wherein panels a and B: cell flow analysis of the effect of the combination of 1 mu M AS2863619 and 5 mu M RO8191 on the erythroid progenitor cell markers CD36, CD71 and erythrocyte marker CD235a on the 15 th day of induced differentiation is carried out, wherein Control is a blank Control group; RO+AS combined action time windows represent Day0-3, day0-6, day3-9, day6-12, day9-12 processing 5 [ mu ] M RO8191 and 1 [ mu ] M AS2863619, respectively, C diagram: cell flow analysis is carried out on the 20 th day of induced differentiation, 1 mu M AS2863619 and 5 mu M RO8191 are combined, different action time windows affect the erythroid progenitor cell marker CD36 and the erythroid marker CD235a, and Control is a blank Control group; RO+AS combined action time windows represent Day0-6, day0-9, day0-12, day0-15, respectively, treated with 5 [ mu ] M RO8191 and 1 [ mu ] M AS2863619 simultaneously.
Detailed Description
The invention establishes a serum-free erythrocyte induced differentiation system with definite chemical components, the differentiation system comprises RO8191 and/or AS2863619, the differentiation system is suitable for the induced differentiation of hematopoietic stem cells or hematopoietic progenitor cells from various sources to erythrocytes, and solves the problems that the induction efficiency of erythroid progenitor cells and erythroid precursor cells is low, the proliferation cannot be effectively carried out, the aging differentiation cannot be carried out quickly and the like in the conventional erythrocyte induced differentiation process, so that the induced differentiation flow of induced pluripotent stem cell-derived hematopoietic stem/progenitor cells (hiPSC-HSPC) to erythrocytes is closer to that of umbilical cord blood-derived hematopoietic stem/progenitor cells (CB-HSPC). The research of the invention discovers that the research of inducing differentiation of hematopoietic stem/progenitor cells from different sources to erythrocytes is greatly promoted, the mass production and preparation of in-vitro erythrocytes are promoted, and the problems of clinical blood shortage and safety are finally solved.
The invention is further illustrated below in conjunction with specific examples, which are intended to illustrate the invention and are not to be construed as limiting the invention. One of ordinary skill in the art can appreciate that: many changes, modifications, substitutions and variations may be made to the embodiments without departing from the spirit and principles of the invention, the scope of which is defined by the claims and their equivalents. The experimental procedure, in which no specific conditions are noted in the examples below, is generally carried out according to conventional conditions or according to the conditions recommended by the manufacturer.
The experimental materials used in the examples of the present invention are shown in table 1 below.
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The experimental methods for detecting cell surface markers by flow cytometry (FACS) according to the embodiments of the present invention are as follows:
1. reagents and antibodies required for FACS detection
(1) Cleaning reagent: buffer A (PBS+4% FBS).
(2) Direct-labeling primary antibody: FITC anti-human CD34 anti-ibody, APC anti-human KDR antibody, PE anti-human PDGFR alpha anti-ibody, PE anti-human CD144 anti-ibody, APC anti-human ITGA3 anti-ibody, PE anti-human EPCR antibody, perCP/cyane 5.5 anti-human CD90 anti-ibody.
2. Preparation of the sample to be tested
(1) Preparing a TrypLE working solution: an appropriate amount of DPBS was pipetted into a new 15 mL centrifuge tube according to 1:1, adding the corresponding volume of TrypLE stock solution, uniformly mixing to obtain working solution, and preheating in a water bath at 37 ℃ for 10 minutes.
(2) Taking differentiated cells from the incubator, sucking off the original culture solution, adding an appropriate amount of DPBS to wash the cells, and washing the cells twice with DPBS (the DPBS dosage is not less than the original culture medium dosage each time), wherein the DBPS is placed in a plate/bottle for 30-45 seconds and sucked out each time for 1 minute.
(3) And adding TrypLE working solution (1 mL TrypLE working solution is added into each hole of a 6-hole culture plate), uniformly covering the bottom of the plate, placing the plate in an incubator for incubation for 2-5 minutes, observing under a microscope during the incubation period, and enabling cells to shrink, round and disperse.
(4) Gently beating the flask/plate to detach the cells from the bottom of the plate, gently beating the plate with a pipette for several times, and finally adding an equal volume of Buffer A to stop digestion, and taking 1×106 cells after cell counting (the suspended cells do not need a cell digestion step, and the suspended cells are directly collected for subsequent operations).
(5) After balancing, centrifuging for 5 min at 200 g, absorbing and discarding the supernatant after centrifugation, flicking the bottom of the centrifuge tube to fully disperse the cells, adding a proper amount of Buffer A for resuspension, centrifuging for 5 min at 200 g, and discarding the supernatant.
(6) Cells were washed 2 times with Buffer a, centrifuged 5 min each for 3 mL Buffer A,200 g, and the supernatant discarded.
(7) Incubating the direct primary antibody: after resuspension of the cells with 100 μl Buffer a, 1 test direct primary antibody was added to each tube, incubated for 30 min at 4 ℃ and flicked the centrifuge tube every 10 min to allow the cells to bind fully with the antibody.
(8) Cells were washed 3 times with Buffer a, centrifuged 5 min each for 3 mL Buffer A,200 g, and the supernatant discarded.
(9) 200 mu L of DPBS (dpBS) resuspended cells are added into each tube, and the cells are filtered through a 70 mu m-pore filter screen to remove undigested cell aggregates, transferred into a 96-hole culture plate, placed at 4 ℃ and preserved in a dark place, and waiting for on-machine detection.
The following is noted: detecting the expression of erythroid progenitor cell marker CD36 and erythroid marker CD235a on 9 th day of induced differentiation; detecting the expression of erythroid progenitor cell markers CD36, CD71 and erythroid marker CD235a on the 12 th day of induced differentiation; detecting the expression of erythroid progenitor cell markers CD36, CD71 and erythroid marker CD235a on 15 th day of induced differentiation; the expression of erythroid progenitor markers CD36, CD71 and erythroid marker CD235a was examined on day20 of induced differentiation.
(10) The method comprises the following steps of stream type on-line detection:
1) The flow cytometer Guava easyCyte HT and computer are turned on.
2) Setting a flow meter; and opening the streaming software and setting various parameters.
3) And after the machine is changed to the Ready state, cleaning the machine.
4) First, the voltages and gains of FSC and SSC were set to place the discrete cell population in the appropriate position in the quadrant, typically with the cell debris in the lower left corner and the larger cell mass in the upper right corner, by isotype control samples. The target cell population is circled, gate is set, and the next analysis is performed.
5) Depending on the antibody-conjugated fluorescein, a suitable detection channel is selected. By adjusting the corresponding channel voltage and compensation, the negative cell population and the positive cell population can be obviously distinguished, and then the experimental samples are sequentially detected.
6) And after the detection is finished, cleaning the flow instrument, and closing the flow instrument and the computer.
Example 1 preparation of Hematopoietic Stem Cells (HSC)
1. Monolayer adherent cell formation
(1) Preparing a TrypLE working solution: sucking 5 mL of DPBS into a new 15 mL centrifuge tube, adding 5 mL of TrypLE stock solution, and uniformly mixing to obtain the TrypLE working solution.
(2) E8 complete medium containing 1% PS (Penicillin-Streptomycin) and 10. Mu. M Y-27632 (ROCKi) was prepared according to the amount of medium required for passage, and 1. Mu. L Y-27632 (10 mM) stock solution was added per ml of medium.
(3) The hiPSC well plate/flask to be passaged was removed from the incubator, the supernatant was aspirated off, and the culture was washed twice with DPBS (DPBS dose no less than the original medium dose) for 1 min each time (DBPS was placed in the well/flask for 30-45 sec and aspirated again at the time of washing).
(4) After TrypLE working solution is added (about 1 mL TrypLE working solution is added to a six-hole plate, about 2 mL TrypLE working solution is added to a T25 bottle), the mixture is placed in an incubator for incubation for 2-5 min, and cells can be observed under a microscope, shrink and become round and disperse.
(5) The flask/plate was gently tapped to detach the cells from the bottom of the plate, then gently swirled several times with a pipette, and finally DMEM/F12 was added to terminate digestion. An appropriate amount of cell suspension was aspirated for counting.
(6) After balancing, centrifuging for 200 g and 5 min, absorbing and removing supernatant after centrifugation, and adding E8 complete culture medium containing 10 mu M Y-27632 for resuspension according to different cell densities at the bottom of a light shake centrifuge tube. After the cells were thoroughly mixed, the cell suspension was dropped into a culture plate well, inoculated cells at a density of 4000 cells/cm2, and placed in a 5% CO2 incubator at 37℃for stationary culture.
(7) After incubation 24 h, the subsequent induced differentiation was performed after washing twice with DPBS.
2. Preparation of HSC induced differentiation medium
Mesoderm induction medium: STEMdiff ™ APEL ™ medium+1% Penicillin-streptomycin+9 μm CHIR99021.
Hematopoietic mesoderm-specific medium: STEMdiff ™ APEL ™ Medium+1% Penicillin-Streptomycin+20 ng/mL VEGF+20 ng/mL bFGF.
Hematopoiesis endothelial specialization and endothelial-hematopoietic cell transformation medium: STEMdiff ™ APEL ™ 2 Medium+1% Penicillium-Streptomycin+20 ng/mL VEGF+20 ng/mL bFGF+20 ng/mL SCF+10 ng/mL IL-3+30 ng/mL TPO+10 ng/mL Flt-L+10 ng/mL BMP4.
3. Mesoendoderm induction (Day 0)
(1) Proper mesoderm induction culture medium is prepared, and the mesoderm induction culture medium is preheated in a water bath kettle at 37 ℃.
(2) The stock culture was aspirated and the cells were washed by adding an appropriate amount of DPBS.
(3) Mesoderm induction medium was added and then placed in a 5% CO2 incubator at 37 ℃ for resting culture 24 h.
4. Hematopoietic mesoderm specialization (Day 1)
(1) Proper amount of hematopoietic mesoderm specific culture medium is prepared, and the culture medium is preheated in a water bath kettle at 37 ℃.
(2) The stock culture was aspirated and the cells were washed by adding an appropriate amount of DPBS.
(3) Hematopoietic mesoderm-specific medium was added and then placed in a 5% CO2 incubator at 37 ℃ for static culture 48 h.
5. Hematopoiesis endothelial specialization and endothelial-hematopoietic cell transformation (Day 3)
(1) Preparing proper amount of hematopoiesis endothelial specialization and endothelial-hematopoietic cell transformation culture medium, and preheating in a water bath at 37 ℃.
(2) After hematopoietic mesoderm specialization 48 h, the stock culture was aspirated; adding a proper amount of DPBS to clean cells, digesting the cells to single cells by using TrypLE working solution, stopping cell digestion, centrifuging for 200 g and 5 min, re-suspending the cells by using a hematopoiesis endothelial specialization and endothelial-hematopoietic cell transformation culture medium, adding 10 mu M Y-27632, and inoculating the cells according to 20000 cells/cm < 2 >; then placing the mixture in a 5% CO2 incubator at 37 ℃ for static culture.
(3) After 24-h culture, fresh hematopoiesis endothelial specialization and endothelial-hematopoietic cell transformation medium were replaced.
(4) Thereafter, fresh hematopoiesis endothelial specialization and endothelial-hematopoietic cell transformation medium was changed every 2 days. Culturing to Day12 and harvesting suspension cells.
EXAMPLE 2 preparation of erythrocytes by differentiation of hematopoietic Stem cells/hematopoietic progenitor cells
The induced differentiation of hematopoietic stem/progenitor cells into erythrocytes involves four phases:
(1) Erythrocyte characterization stage (Day 0-6)
The cell density is: 1X 105/mL;
the culture medium comprises the following components: IMDM, 1% ITS-X, 50 μg/mL Ascorbic Acid (AA), 50 μM Acetylcysteine (NAC), 50 μM quinine dimethacrylate (Trolox), 0.5% BSA (bovine serum albumin), 50 ng/mL SCF (stem cell growth factor), 10 ng/mL IL-3 (interleukin-3), 10 ng/mL EPO (erythropoietin), 1 μM Dexamethasone (Dexamethasone).
(2) Erythrocyte expansion stage (Day 6-12)
The cell density is: 5X 105/mL;
the culture medium comprises the following components: IMDM, 1% ITS-X, 50 [ mu ] g/mL ascorbic acid, 50 [ mu ] M acetylcysteine, 50 [ mu ] M quiniodimethacrylate, 0.5% BSA, 50 ng/mL SCF, 10 ng/mL IL-3, 10 ng/mL EPO, 1 [ mu ] M Dexamethasone.
(3) Erythrocyte maturation stage (Day 12-15)
The cell density is: 1X 106/mL;
the culture medium comprises the following components: IMDM, 1% ITS-X, 50 μg/mL ascorbic acid, 50 μM acetylcysteine, 50 μM quinine dimethacrylate, 0.5% BSA, 50 ng/mL SCF, 10 ng/mL EPO.
(4) Erythrocyte enucleation stage (Day 15-20)
The cell density is: 1X 106/mL;
the culture medium comprises the following components: IMDM, 1% ITS-X, 50 μg/mL ascorbic acid, 50 μM acetylcysteine, 50 μM quinine dimethacrylate, 0.5% BSA, 10 ng/mL EPO.
During erythrocyte induced differentiation, fresh medium was changed every 2 days until Day20.
Example 3 Effect of Garcinone D, RO8191 and AS2863619 on erythroid progenitor or erythroid precursor cell production 1
1. Experimental method
The effect of Garcinone D (GD), RO8191 (RO) and AS2863619 (AS) on erythroid progenitor or erythroid precursor induced differentiation was tested separately.
The experimental grouping is as follows: control group-blank Control group (processing mode is the same AS example 2), gd+as+ro group (5 μm Garcinone D, 5 μm RO8191 and 1 μm AS2863619 are added simultaneously during Day0-12 in example 2). Flow assays were performed on day12 of differentiation.
2. Experimental results
The results of the cell flow analysis are shown in FIG. 1A, and the results show that the addition of 5 mu M Garcinone D, 5 mu M RO8191 and 1 mu M AS2863619 (GD+AS+RO group) significantly promote the induced differentiation of CD36+ cells during the 0 th to 12 th days of the induced differentiation of erythrocytes, wherein the induced efficiency of CD36+CD233a-erythroid progenitor cells is 35.18% and the induced efficiency of CD36+CD233a+ erythroid precursor cells is 54.82%; whereas the Control group (Control) was obtained only with 0.85% CD36+CD235a-erythroid progenitor cells, 0.88% CD36+CD235a+ erythroid precursor cells, 45.26% CD235a+CD36-erythrocytes. Meanwhile, the results showed that Garcinone D, RO8191 and AS2863619 significantly promoted the production of cd71++ cells. The results of the cell morphology change are shown in FIG. 1B, which shows that Garcinone D, RO8191 and AS2863619 can promote the generation of cell colonies. The above results indicate that Garcinone D, RO8191 and AS2863619 are capable of promoting differentiation and proliferation of cd36+cd71++ erythroid progenitor cells or erythroid precursor cells.
Example 4 Effect of Garcinone D, RO8191 and AS2863619 on erythroid progenitor or erythroid precursor cell production 2
1. Experimental method
To further determine the specific effects of Garcinone D, RO8191 and AS2863619, this example tested the effects of the above 3 agents alone and in combination on erythrocyte induced differentiation, respectively. On days 0-12 of erythrocyte induced differentiation (days 0-12 described in example 2), 5 μm Garcinone D (GD), 5 μm RO8191 (RO), 1 μm AS2863619 (AS) or 5 μm RO8191 and 1 μm AS2863619 (ro+as) were added, respectively, and flow analysis was performed on days 9 and 15 of induced differentiation, respectively.
Experimental grouping: control-blank group, GD-Day 0-12 is treated with 5 [ mu ] M Garcinone D, RO-Day 0-12 is treated with 5 [ mu ] M RO8191, AS-Day 0-12 is treated with 1 [ mu ] M AS2863619, RO+AS-Day 0-12 is treated with 5 [ mu ] M RO8191 and 1 [ mu ] M AS2863619 simultaneously.
2. Experimental results
The results of the induced differentiation day9 cell flow assay are shown in FIG. 2A, which shows that both RO8191 and AS2863619 promote the production of CD36+CD233a-erythroid progenitor cells (17.93%, 26.93%) and CD36+CD233a+ erythroid precursor cells (3.48%, 10.62%), and are significantly better than Garcinone D. Whereas RO8191 and AS2863619 synergistically significantly enhance the production of both cd36+cd235a-erythroid progenitor cells (22.08%) and cd36+cd235a+ erythroid precursor cells (43.61%), i.e. the combination of both RO8191 and AS2863619 produces a synergistic effect.
Meanwhile, the invention discovers that the combination of RO8191 and AS2863619 can obviously improve the expression of erythroid progenitor cells and erythroid precursor cell surface markers CD71, and the synergistic effect of RO8191 and AS2863619 greatly promotes the generation of CD71++ cells (66.23% vs. 16.10% or 39.63%) compared with the independent effect of RO8191 and AS2863619 (see figure 2A), and the result again proves that the combination of RO8191 and AS2863619 has the synergistic effect.
The results of the induced differentiation day15 cell flow analysis are shown in FIG. 2B, and the results show that the differentiation efficiency of the CD36+ cells in the AS2863619 treatment group is 62.52% and the differentiation efficiency of the CD36+ cells in the AS2863619+RO8191 treatment group is 78.25%; whereas the Control and RO8191 treatment groups have CD36+ cell differentiation efficiencies of only 6.43% and 10.28%, respectively.
The experimental results show that AS2863619 is a key factor for promoting the expression of CD36+ cells; and the joint action of RO8191 and AS2863619 activates JAK/STAT signal paths to obviously promote the generation of CD36+ and CD71+ erythroid progenitor cells and erythroid precursor cells, so that the differentiation process of hiPSC-HSPC to erythrocytes is closer to CB-HSPC, and the problems that the erythroid progenitor cells and erythroid precursor cells are low in generation efficiency and the expansion of erythroid progenitor cells and erythroid precursor cells cannot be effectively realized in the differentiation process of hiPSC-HSPC to erythrocytes are solved.
Example 5 time window for the co-operation of roup 8191 with AS2863619 to promote erythroid progenitor and erythroid precursor cell production grope 1
1. Experimental method
To further explore the optimal time window of action of RO8191 in conjunction with AS2863619 to promote the production of CD36+ and CD71+ erythroid progenitors and erythroid precursors, this example additionally added 5 [ mu ] M RO8191 on days 0-12 (days 0-12 described in example 2) on days 0-3 (D0-3), days 0-6 (D0-6), days 0-9 (D0-9) and days 0-12 (D0-12) of induced differentiation, respectively, on day15 of differentiation.
Experimental grouping: control-Control, AS-Day 0-12 was treated with 1 [ mu ] M AS2863619, AS+D0-3 RO-was treated with 5 [ mu ] M RO8191 on the basis of Day0-3 on the basis of Day0-12 being treated with 1 [ mu ] M AS2863619, AS+D0-6 RO-was treated with 5 [ mu ] M RO8191 on the basis of Day0-6 on the basis of Day0-12 being treated with 1 [ mu ] M AS2863619, AS+D0-9 RO-was treated with 5 [ mu ] M RO8191 on the basis of Day0-12 being treated with 1 [ mu ] M AS2863619, AS+D0-12 RO-was treated with 5 [ mu ] M RO8191 on the basis of Day0-12 being treated with 1 [ mu ] M AS2863619.
2. Experimental results
The results show that AS2863619 treatment significantly improved the induction efficiency of cd71+cd235a+ (. Gtoreq. 49.18%) and cd36+cd235a+ (. Gtoreq.21.19%) erythroid precursor cells relative to Control; on the basis of AS2863619, the addition of RO8191 obviously improves the induction efficiency of CD71+CD235a+ (. Gtoreq. 63.77%) and CD36+CD23235a+ (. Gtoreq. 25.77%) erythroid precursor cells. Meanwhile, the present invention found that the longer RO8191 treatments of D0-9 and D0-12 are more advantageous for maintaining CD36+ cell production and maintenance than the shorter RO8191 treatments of D0-3 and D0-6 (see FIG. 3). The above results indicate that RO8191 promotes induced differentiation of cd71+cd235a+ and cd36+cd235a+ erythroid precursor cells in cooperation with AS2863619, and that the duration of action of RO8191 is an important factor for maintaining cd36+ cell production and maintenance.
Example 6 time window for the co-operation of R8191 with AS2863619 to promote erythroid progenitor and erythroid precursor cell production 2
1. Experimental method
To further understand the point in time when RO8191 and AS2863619 co-operate, this example explored the window of time when RO8191 and AS2863619 co-operate. 1. Mu.M AS2863619 and 5. Mu.M RO8191 are added at the same time on day0-3 (D0-3), day0-6 (D0-6), day0-9 (D0-9), day0-12 (D0-12), day0-15 (D0-15), day3-6 (D3-6), day3-9 (D3-9), day6-9 (D6-9), day6-12 (D6-12), and day9-12 (D9-12) of induced differentiation, and flow detection is performed on day15 of differentiation.
2. Experimental results
The results show that the synergy of RO8191 and AS2863619 at early stages of D0-3, D0-6, D3-6 and D3-9 significantly increases the differentiation efficiency of CD36+CD233a+ erythroid precursor cells relative to the Control, and the earlier and longer the action time, the higher the differentiation efficiency of CD36+CD233a+ erythroid precursor cells; whereas the synergy of RO8191 and AS2863619 at the late stages of D6-9, D6-12 and D9-12 does not effectively increase the differentiation efficiency of CD36+CD235a+ erythroid precursor cells; however, the synergistic effect of RO8191 and AS2863619 effectively increases the induction efficiency of CD71++ CD235a+ erythroid precursor cells (see FIGS. 4A and 4B). The above results indicate that the effective time window for the synergistic effect of RO8191 and AS2863619 to promote differentiation of cd36+cd235a+ erythroid precursor cells is located at the early stage of erythroid induced differentiation. Subsequently, the present example was searched for a longer action time window at early stage, and the results of the flow analysis on day20 showed that treatment of RO8191 and AS2863619 with D0-6, D0-9, D0-12 and D0-15 significantly improved the CD36+ cell differentiation efficiency (72.74% vs. 87.66%, 91.86%, 93.9%, 94.28%) relative to the control group; however, more of the cells in the D0-6 treated group were CD36+CD235a+ erythroid precursor cells, while the longer treated group had a higher proportion of CD36+CD235a-erythroid precursor cells (see FIG. 4C). The above results indicate that the synergistic effect of RO8191 and AS2863619 at early and long-term differentiation induction is more advantageous for enhancing the induction of differentiation of erythroid progenitor cells and erythroid precursor cells.
The above description of the embodiments is only for the understanding of the method of the present invention and its core ideas. It should be noted that it will be apparent to those skilled in the art that several improvements and modifications can be made to the present invention without departing from the principle of the invention, and these improvements and modifications will fall within the scope of the claims of the invention.

Claims (10)

  1. Use of rou8191 and/or AS2863619 for promoting differentiation and proliferation of erythroid progenitor cells or erythroid precursor cells.
  2. 2. A basal medium for promoting the induced differentiation of hematopoietic stem cells or hematopoietic progenitor cells into erythroid progenitor cells or erythroid precursor cells, wherein the basal medium comprises RO8191 and/or AS2863619.
  3. 3. The basal medium of claim 2, further comprising IMDM medium, ITS-X, ascorbic acid, acetylcysteine, quinine dimethacrylate, BSA.
  4. 4. A basal medium according to claim 3, wherein the basal medium comprises the following components: 5. mu M RO8191, 1 mu M AS2863619, 1% ITS-X, 50 mu g/mL ascorbic acid, 50 mu M acetylcysteine, 50 mu M quinio dimethacrylate, 0.5% BSA.
  5. 5. A culture system for promoting the induction and differentiation of hematopoietic stem cells or hematopoietic progenitor cells into erythroid progenitor cells or erythroid precursor cells, characterized in that the culture system comprises a erythroid specification stage culture medium, a erythroid expansion stage culture medium and a erythroid maturation stage culture medium;
    the erythrocyte differentiation stage medium comprising the basal medium, SCF, IL-3, EPO, dexamethasone according to any one of claims 2-4;
    the erythrocyte expansion stage medium comprising the basal medium of any one of claims 2-4, SCF, IL-3, EPO, dexamethasone;
    the erythrocyte maturation stage medium comprises the basal medium, SCF, EPO of any of claims 2-4.
  6. 6. The culture system according to claim 5, wherein the content of each component in the erythrocyte-specific stage medium is: 50 ng/mL SCF, 10 ng/mL IL-3, 10 ng/mL EPO, 1 [ mu ] M dexamethasone;
    the erythrocyte expansion stage culture medium comprises the following components in percentage by weight: 50 ng/mL SCF, 10 ng/mL IL-3, 10 ng/mL EPO, 1 [ mu ] M dexamethasone;
    the content of each component in the culture medium at the erythrocyte maturation stage is respectively as follows: 50 ng/mL SCF, 10 ng/mL EPO.
  7. 7. A method of promoting the induced differentiation of hematopoietic stem or progenitor cells into erythroid progenitor cells or erythroid precursor cells, comprising culturing the hematopoietic stem or progenitor cells using the culture system of claim 5 or 6.
  8. 8. The method according to claim 7, characterized in that it comprises the steps of:
    (1) Day0-6, a erythrocyte-specific stage, culturing hematopoietic stem cells or hematopoietic progenitor cells using the erythrocyte-specific stage medium of claim 5 or 6;
    (2) Day6-12, a erythrocyte expansion stage, culturing the cells obtained in step (1) using the erythrocyte expansion stage medium of claim 5 or 6;
    (3) Day12-15, culturing the cells obtained in step (2) with the erythroid progenitor cells or erythroid precursor cells according to claim 5 or 6.
  9. 9. The method of claim 8, wherein the medium used during Day0-3, day0-6, day0-9, day0-12, or Day0-15 contains RO8191 and AS2863619.
  10. 10. The method of claim 8, wherein the cell density in step (1) is 1X 10 5 individual/mL;
    cell density in step (2)Is 5 multiplied by 10 5 individual/mL;
    the cell density in step (3) was 1X 10 6 And each mL.
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