Serum-free feeder-layer-free method for efficiently inducing directional differentiation and maturation of human hematopoietic stem cell erythroid

Abstract

The invention relates to a method for efficiently inducing human hematopoietic stem progenitor cell erythroid to be directionally differentiated into mature erythrocytes in a serum-free and feeder-layer-free manner, which adopts a serum-free and feeder-layer-cell-free induction culture system, and uses an mTOR pathway inhibitor rapamycin to regulate the activity of an mTOR pathway in the erythroid differentiation process. The use dosage and the treatment mode of the mTOR pathway inhibitor rapamycin are further optimized, and the erythrocyte denucleation efficiency of an in-vitro induced differentiation system is remarkably improved to 70% under the condition that erythrocyte proliferation and differentiation are not affected or the cell state is not reduced. According to the method, heterologous pollution caused by animal serum or heterologous cells is effectively avoided, the clinical safety of in-vitro preparation of transfusion-level red blood cells in the future is ensured, the erythroid directional induced differentiation final maturation efficiency of the human hematopoietic stem progenitor cells is improved, meanwhile, the reagent cost is reduced, and large-scale preparation and clinical transformation are facilitated.

Description

Technical Field

[0001] The invention belongs to the field of biopharmaceuticals, and specifically relates to a method for efficiently inducing erythroid directional differentiation and maturation of human hematopoietic stem cells without serum or feeder layers. Background Art

[0002] In order to find a more sufficient, safe and economical blood substitute source besides blood donation volunteers and solve the problem of clinical blood transfusion sources, researchers have made attempts and efforts in many fields. In the field of chemical synthesis, although red blood cell substitutes including hemoglobin oxygen carriers HBOCs and fluorocarbon compounds PFCOCs have been developed, they cannot be used in clinical practice due to their defects in stability, cost, oxygen carrying capacity and toxic side effects.

[0003] With the progress of stem cell research and technology, it has become possible to use hematopoietic stem cells to culture in vitro to prepare mature red blood cells as an alternative source for blood transfusion. To date, many research groups have successfully induced the production of reticulocytes in vitro from human hematopoietic stem cells from different sources. The main ideas for inducing the preparation of red blood cells in vitro are as follows: one is to induce differentiation to obtain red blood cells by co-culturing with stromal cells derived from hematopoietic tissue as a feeder layer; the other is to induce pluripotent stem cells / hematopoietic stem cells to differentiate into red blood cells by adding various growth factors at different stages of induction of differentiation. The cell culture system of this strategy can be divided into two culture systems: containing animal serum and without animal serum. (1) Relevant progress of co-culture scheme: Currently, the commonly used stromal cells in co-culture schemes include mouse stromal cell lines such as OP9, S17, and MS5. Choi et al. found that the stromal cell OP9 can induce human ES cells and iPSc to differentiate into hematopoietic and erythroid cells through co-culture. Vodyanik et al. obtained CD34+ cells from hESCs by co-culturing with OP9 without adding any factors. However, the obtained cells did not express the pan-leukocyte marker (CD45), indicating that co-culture with OP9 cells reproduced the early stages of erythropoiesis. Similarly, Kaufman et al. obtained multiple hematopoietic cell chains after several days of culture by co-culturing hESCs (H1, H1.1 and H9.2 lines) without any factors with the mouse stromal cell line S17 or the mouse yolk sac endothelial cell line C166. Giarratana et al. developed a method for large-scale in vitro production of mature erythrocytes from HSPCs of umbilical cord blood and peripheral blood by co-culturing with mouse MS-5 cells. This method can achieve up to 2 million-fold expansion of the number of denuclearized erythrocytes, and the obtained ex vivo erythrocytes are nearly fully (90%) mature. In addition, co-culture with mouse fetal liver-derived stromal cells (mFLSCs) can also obtain mature erythrocytes expressing β-globin and denucleated from hESCs, which can achieve hemoglobin conversion and oxygen transport. There are also several research groups that have used human-derived stromal cells such as human fetal liver stromal cells and human mesenchymal stem cells to co-culture with human hematopoietic stem and progenitor cells to obtain mature red blood cells. Baek et al. used human umbilical cord blood or bone marrow-derived mesenchymal stem cells (MSCs) as a differentiation program for feeder cells, with a denuclearization rate of up to 64%. Fujimi et al. used umbilical cord blood-derived HSPCs co-cultured with human macrophages to induce red blood cell differentiation, avoiding xenogeneic feeding. This process can produce 1.76×10 hematopoietic stem cells from 1.0 unit CB. 13(8.8 units) of red blood cells, with a denucleation rate of 99.4%. Magin compared three feeder cells for co-culture of human cord blood hematopoietic stem cells, namely mesenchymal stem cells (MSC), human umbilical vein endothelial cells (HUVEC) and Wharton's jelly cells (WJC), and found that WJC was the best choice, and MSC was better than HUVEC. (2) Directed induction scheme without trophoblast cells: The induction scheme without co-culture of feeder cells can reduce the complexity of the overall operation and is suitable for expansion using existing bioreactor technology, which is conducive to future large-scale preparation. This type of scheme mainly uses a combination of various growth factors to simulate the microenvironment of each differentiation stage in the process of erythropoiesis, and directs the differentiation of hematopoietic stem and progenitor cells into erythrocytes. The main growth factors are stem cell factor (SCF), interleukin-(IL-) 3, IL-6, and erythropoietin (EPO). At present, a variety of induction schemes have been established, and the in vitro induction culture system of erythrocytes has also been continuously optimized.

[0004] Douay et al. used a feeder-free culture system containing 5% human inactivated plasma combined with growth factors to induce the differentiation of hematopoietic stem progenitor cells (HSPCs) isolated from adult peripheral blood into red blood cells, with an enucleation rate of 80%. The differentiated cRBCs were injected into immunodeficient mice and donor patients. About 50% of the reticulocytes could be detected in the peripheral blood within 26 days after transplantation, confirming for the first time that red blood cells differentiated in vitro have physiological functions.

[0005] In an attempt to scale up the laboratory red blood cell culture method to a bioreactor system for large-scale preparation, Zhang et al. used a spinner bottle culture system to culture 5×10 red blood cells from umbilical cord blood in the presence of 15% fetal bovine serum. 6 2×10 CD34+ hematopoietic stem and progenitor cells were induced 8 The percentage of erythrocytes with denucleation reached 50%±5.7%. Timmins et al. used a serum-free induction system to induce erythrocyte differentiation of CD34+ hematopoietic stem cells from umbilical cord blood through a rolling stirred bioreactor, reaching 1.7x 10 6 times higher amplification efficiency.

[0006] Neildez-Nguyen et al. used a serum-free induction system to expand 20,000 times of erythroid precursor cells (equivalent to approximately 0.5 transfusion units) from CD34+ cells derived from umbilical cord blood, but the enucleation rate was extremely low. Miharada et al. used an induction scheme that was serum-free and feeder-free, containing 5% human plasma protein components and high-concentration transferrin, and successfully induced erythroid differentiation of CD34+ hematopoietic stem and progenitor cells derived from umbilical cord blood, with an enucleation rate of up to 70%, but the reagent cost was extremely expensive.

[0007] In summary, the technology of preparing red blood cells by in vitro induced differentiation of hematopoietic stem cells has made great progress. The corresponding induced differentiation system, cell purification and evaluation process have been established. There are certain differences among various schemes. However, in order to simulate the hematopoietic microenvironment of red blood cell differentiation in vivo as much as possible during the in vitro differentiation process, improve the expansion efficiency and denucleation efficiency of induced differentiation of red blood cells, and achieve the goal of in vitro large-scale preparation of clinical transfusion-grade red blood cells, the following problems still exist: (1) The main induction processes and factors used are basically similar or identical, with only a few differences in culture medium components and the presence or absence of feeder cell co-culture; (2) One type of induced differentiation scheme currently still uses natural additives, namely fetal bovine serum. or human AB type serum, so there is a risk of animal or human pathogens contaminating the culture system, thus posing a safety issue; (3) At present, there are still many culture systems that use co-culture with mouse or human stromal cells, which are contaminated by animal components and increase the complexity of the operation, making it impossible to prepare on a large scale; (4) The low efficiency of terminal enucleation is still a key obstacle that needs to be overcome in the preparation of in vitro red blood cells. At present, the red blood cell enucleation efficiency of most serum-free and feeder-free in vitro induced differentiation systems is about 5-10%; (5) The current induction system for in vitro preparation of red blood cells, especially the serum-free system, needs to achieve a relatively high enucleation efficiency, and its reagents and other costs are very expensive, making it difficult to use for large-scale production. Summary of the invention

[0008] The purpose of the present invention is to provide a method for efficiently inducing erythroid differentiation and maturation of human hematopoietic stem and progenitor cells without serum or feeder layer, so as to solve the existing technical problems. In order to ensure the safety of preparing transfusion-grade red blood cells in vitro for clinical use in the future, the present invention adopts a serum-free, chemically clear, feeder-free system, and improves the efficiency of terminal maturation of erythroid directional differentiation of human hematopoietic stem and progenitor cells by regulating the activity of the mTOR pathway, while greatly reducing the reagent cost under the premise of achieving a high denucleation efficiency.

[0009] A method for efficiently inducing erythroid-directed differentiation and maturation of human hematopoietic stem and progenitor cells without serum or feeder layer, comprising inducing and culturing CD34+ hematopoietic stem and progenitor cells in vitro with erythrocyte induction differentiation fluid, and then using mTOR pathway inhibitor rapamycin to regulate the activity of mTOR pathway during erythroid differentiation.

[0010] Further, the following steps are included: (1) Resuspend CD34+ hematopoietic stem and progenitor cells in erythrocyte differentiation medium 1 and culture them in 5% O2; (2) Collect the cells obtained in step (1), centrifuge, remove the supernatant, and resuspend them in red blood cell induction differentiation solution 2; determine whether to plate them according to the cell density; the red blood cells cultured in the present invention grow in suspension, and determine whether to plate them according to the cell counting results. The cell density is generally not higher than 1×10 6 cells / ml, that is, no more than 1 million cells per ml; (3) Collecting the cells obtained in step (2), centrifuging them, removing the supernatant, and resuspending them in red blood cell induction differentiation solution 3; (4) using 25-50 nM of the mTOR pathway inhibitor rapamycin to regulate the mTOR pathway activity of the cells obtained in step (3), and continuing the treatment for 8-14 days; Among them, erythroid induction differentiation fluid 1 is a basic induction differentiation fluid with the addition of cytokines EPO, IL-3 and SCF; erythroid induction differentiation fluid 2 is a basic induction differentiation fluid with the addition of cytokines EPO and SCF; erythroid induction differentiation fluid 3 is a basic induction differentiation fluid with the addition of cytokine EPO.

[0011] Furthermore, step (4) is to use 25 nM of the mTOR pathway inhibitor rapamycin after the 14th day of in vitro induction.

[0012] Furthermore, the formula of the basic induction differentiation solution is as follows: Glutamate 4mmol / L, Penicillin 100 U / mL, Streptomycin 100 μg / mL, Inositol 4 μg / mL, Folic acid 10 μg / mL, Ferrous sulfate 0.9 μg / mL, Ferric nitrate 0.09 μg / mL, Human transferrin 150μg / mL, Recombinant human serum albumin 5% (50g), Trypsin inhibitor 1ng / mL, Sodium selenite 6.7ng / mL, Reduced glutathione 1 μg / mL, Arachidonic acid 2ng / mL, Cholesterol 220ng / mL, Linoleic acid 10ng / mL, Linolenic acid 10 ng / mL, Oleic acid 10 ng / mL, Palmitoleic acid 10ng / mL, Heparin 5 U / mL Make up to 1L with IMDM.

[0013] Furthermore, the culture time in step (1) is 7 days, and the liquid is replaced every 2 to 3 days during the culture period.

[0014] Furthermore, after the mTOR pathway inhibitor rapamycin is added in step (4), fresh basal induction differentiation medium containing corresponding concentration of rapamycin is replaced every 2 to 3 days.

[0015] The erythrocytes derived from the differentiation of human hematopoietic stem and progenitor cells obtained by the above method.

[0016] The present invention uses the mTOR pathway inhibitor rapamycin to regulate the activity of the mTOR pathway during erythroid differentiation, and compares and optimizes the time, timing, concentration and intervention treatment method of rapamycin treatment to select the minimum effective dose and action time to improve the terminal maturation efficiency of erythrocytes. Specifically, firstly, based on the data of continuous exploration and analysis of the treatment timing, it is determined that the best time point is to perform different treatments on the cells starting from the 14th day of erythrocyte induction differentiation to observe whether it affects the differentiation process and the final differentiation and maturation efficiency; then, according to the concentration and treatment method of rapamycin, the following groups are divided: the rapamycin dose is divided into three final concentrations of 25nM, 50nM, and 75nM (i.e., high, medium, and low concentrations); the treatment method is divided into three methods: continuous treatment, intermittent treatment (i.e., starting from 14 days of induction, adding drugs for 3 days, withdrawing drugs for 3 days, and so on in a cycle), and short-term treatment (stopping the drug after 3 days of treatment).

[0017] Compared with the prior art, the serum-free, chemically defined, and feeder-free induction culture system established by the present invention can effectively avoid heterologous contamination caused by animal serum or xenogeneic cells, and can be used for in-depth research on the mechanism of erythroid differentiation, establishment of disease models, and drug screening. At the same time, under the condition of relatively low reagent costs, the present invention greatly improves the efficiency of terminal denuclearization and maturation of directional differentiation of hematopoietic stem and progenitor cells in vitro, which is an attempt to induce the production of clinical transfusion-grade erythrocytes in vitro, and also provides a certain methodological basis for the future large-scale preparation and clinical transformation. It has the following advantages: (1) The culture system of the present invention removes human or animal components and adopts a serum-free, chemically defined, feeder cell-free induction culture system, thereby reducing the risk of introduction of exogenous contamination sources and avoiding potential biological and clinical safety risks in future transformation processes; it solves the problem of animal or human pathogen contamination and allergens caused by the use of animal / human serum or matrix cell co-culture in some current solutions.

[0018] (2) The present invention uses a pathway inhibitor to improve the efficiency of terminal denucleation of erythrocytes. By using the clinically approved drug rapamycin to regulate the activity of the mTOR pathway, the efficiency of erythrocyte denucleation in the in vitro induced differentiation system is significantly improved, reaching 50% on the 28th day of induction. If the induction time is further extended to 40 days, the maximum denucleation efficiency can reach 70%. This solves the problem of low efficiency of terminal denucleation of erythrocytes induced in vitro in vitro induction in the existing serum-free culture system.

[0019] (3) The present invention controls the reagent cost required for inducing red blood cell differentiation and uses relatively low-cost drugs to promote cell maturation efficiency, which greatly reduces the reagent cost and achieves good results, laying a solid foundation for future large-scale production and commercial transformation.

[0020] (4) The present invention further optimizes the dosage and treatment method of the mTOR pathway inhibitor rapamycin, and significantly improves the efficiency of denuclearization and maturation of erythrocytes at the terminal stage of differentiation without affecting erythrocyte proliferation and differentiation or reducing the cell state. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 Flow chart of the optimization scheme for directed induced differentiation of red blood cells of the present invention.

[0022] Figure 2 (A) shows the dynamic changes of cell surface antigens CD235a and CD71 detected by flow cytometry at different time points during the erythroid differentiation induction stage; (B) shows the statistical results of the percentage of the corresponding surface antigens.

[0023] Figure 3 This is a quantitative statistical analysis of the changes in the expression levels of erythrocyte-specific globin genes (A) and erythrocyte differentiation-related genes (B) at different time points.

[0024] Figure 4 (A) shows the flow cytometry detection of β-, γ-, ε- and other types of globin in induced erythrocytes after immunofluorescence staining on the 16th day of the second stage of cell differentiation; (B) shows the quantitative statistical analysis results of the percentage of cells expressing each type of globin.

[0025] Figure 5 (A) and (B) are the cell enucleation efficiency and the percentage of enucleated cells in different treatments at a final concentration of 75 nM rapamycin, respectively; (C) and (D) are the cell enucleation efficiency and the percentage of enucleated cells in different treatments at a final concentration of 50 nM rapamycin, respectively; (E) and (F) are the cell enucleation efficiency and the percentage of enucleated cells in different treatments at a final concentration of 25 nM rapamycin, respectively.

[0026] Figure 6(A) and (B) are the cell enucleation efficiency and the percentage of enucleated cells after treatment with different concentrations of rapamycin under the intervention mode of continuous treatment, respectively; (C) and (D) are the cell enucleation efficiency and the percentage of enucleated cells after treatment with different concentrations of rapamycin under the intervention mode of intermittent treatment, respectively; (E) and (F) are the cell enucleation efficiency and the percentage of enucleated cells after treatment with different concentrations of rapamycin under the intervention mode of stopping treatment after 3 days.

[0027] Figure 7 The three groups treated with rapamycin continuously, including 75nM, 50nM, and 25nM, were stained with Wright-Giemsa on day 32 to observe and compare the cell status and denucleation from a morphological perspective. The red arrows indicate pink denucleated mature erythrocytes, and the blue arrows indicate non-denucleated erythrocytes (the nucleus appears blue).

[0028] Figure 8 (A) and (B) show the effects of rapamycin treatment with a final concentration of 50 nM (intermediate concentration) on the formation of erythroid burst colonies (BFU-E) and erythroid colony-forming units (CFU-E) starting from day 1 of differentiation, respectively.

[0029] Fig. 9 (A), (B), and (C) are the cell growth curves after rapamycin treatment on days 8, 11, and 14 of erythroid differentiation, respectively.

[0030] Fig.10 (A), (B), and (C) show the expression of erythrocyte-specific surface antigens CD71 and CD235a detected by flow cytometry at different time points after rapamycin treatment on days 8, 11, and 14 of erythroid differentiation.

[0031] Fig.11 The cell cycle was detected by flow cytometry after PI staining and rapamycin treatment on day 8 and day 11 of erythroid differentiation (A), and the cell proliferation was detected by flow cytometry after CFDA-SE staining (B).

[0032] Fig.12 The cell proliferation and cell cycle were detected by flow cytometry using BrdU staining after rapamycin treatment on days 8 and 11 of erythroid differentiation.

[0033] Fig.13 Annexin V staining and flow cytometry were used to detect cell apoptosis after rapamycin treatment on the 8th and 11th days of erythroid differentiation. DETAILED DESCRIPTION

[0034] In order to make the objectives, technical solutions and advantages of the present invention more clear, the present invention will be further described in detail below with reference to the accompanying drawings.

[0035] Example 1 A method for efficiently inducing erythroid differentiation and maturation of human hematopoietic stem and progenitor cells without serum or feeder layer, such as Figure 1 As shown, the following steps are included: 1. Directed differentiation of hematopoietic stem cells into erythrocytes 1) Erythroid differentiation induction day 0 to 8 ① Prepare the erythroid differentiation induction medium 1 (day 0 to 8) as shown, that is, add the factor 1 shown in Table 2 to the basic differentiation induction medium shown in Table 1.

[0036]

[0037]

[0038] ② CD34+ hematopoietic stem and progenitor cells were resuspended in the red blood cell induction differentiation solution 1 (days 0 to 8) prepared in step ① and cultured under 5% O2 conditions; ③ During this stage, the cells are cultured with red blood cell induction differentiation solution 1 (day 0-8) from day 0 to day 8, and the solution is changed every 2-3 days. Generally, obvious proliferating colonies can be observed around day 4, and the proliferation rate increases significantly around day 7; ④ On the 8th day, flow cytometry was performed to detect the expression of red blood cell differentiation-related surface antigens, including CD235a, CD71, CD44, etc. Several detection time points such as the 2nd day, 4th day, and 6th day can be added according to the number of cells.

[0039] 2) Erythroid differentiation induction day 8 to 14 ① Prepare red blood cell induction differentiation solution 2 (Day 8-14): add factor 2 shown in Table 3 to the basic induction differentiation solution shown in Table 1;

[0040] ② On the 8th day, collect the cells in a 15mL centrifuge tube, centrifuge at 1200r / min for 5min, remove the supernatant and resuspend in red blood cell induction differentiation medium 2 (8th to 14th day); determine whether to split the cells based on the cell density. Generally, splitting the cells is required on the 8th and 11th days to reduce the cell density.

[0041] ③ On the 12th day, a small amount of cells were taken for flow cytometry analysis.

[0042] 3) After 14 days of erythroid differentiation ① On the 14th day, collect the cells in a 15mL centrifuge tube, centrifuge at 1200r / min for 5min, remove the supernatant and resuspend in erythrocyte induction differentiation solution 3 (14 days~); after the 14th day, the cell proliferation rate gradually decreases, and the interval between changing the solution can be extended to 3 to 4 days, but the liquid should not be too little to prevent evaporation. Preparation of erythrocyte induction differentiation solution 3 (14 days~): add factor 3 shown in Table 4 to the basic induction differentiation solution shown in Table 1;

[0043] ② On the 16th day, a small amount of cells were taken for flow cytometry analysis.

[0044] ③ Then, cells were collected every 4 days for flow cytometry analysis.

[0045] 2. Optimization of rapamycin treatment and dosage during erythroid differentiation This experiment started to treat the cells differently on the 14th day of induced differentiation of red blood cells, and finally observed whether it affected the differentiation process and the final differentiation and maturation efficiency. According to the amount of rapamycin used and the treatment method, a total of 9 groups were divided: the rapamycin dose was divided into three final concentrations of 25nM, 50nM, and 75nM (to ensure the consistency of the concentration of the added solvent DMSO, three concentrations of rapamycin storage solution of 25μM, 50μM, and 75μM were prepared, 1000×); the treatment method was divided into three methods: continuous treatment, intermittent treatment, and short-term treatment (stop after 3 days of treatment); plus the DMSO solvent group and the blank control group, a total of 11 groups. The specific operations are as follows: 1) Blank control group: This group did not receive any intervention treatment, and used erythroid induction medium to induce the directed differentiation of hematopoietic stem and progenitor cells.

[0046] 2) Solvent group: DMSO was added to the erythrocyte differentiation induction solution at 1 μL / mL (the same amount as 1000× rapamycin added to the treatment group) to eliminate the influence of the solvent on the experimental results, and other induction.

[0047] 3) 25nM rapamycin: divided into three groups according to different treatment methods, namely: ① Continuous treatment group: rapamycin was added to the erythrocyte differentiation medium at a final concentration of 25 nM, and the rest of the operations remained unchanged.

[0048] ② Intermittent treatment group: Rapamycin was added to the erythrocyte induction differentiation medium at a final concentration of 25 nM. The differentiation medium containing rapamycin was added on days 14-17, 20-23, and 26-29, respectively. The cells were cultured with differentiation medium without rapamycin in between.

[0049] ③ Short-term treatment group: rapamycin was added to the erythrocyte differentiation medium at a final concentration of 25 nM. The differentiation medium containing rapamycin was added on days 14-17, and no rapamycin was added at other times.

[0050] The continuous treatment group was replaced with fresh induction differentiation medium containing corresponding concentration of rapamycin every 2 to 3 days. The intermittent treatment and short-term treatment groups were washed once with 1×PBS or serum-free culture medium before replacing fresh induction differentiation medium to remove residual rapamycin as much as possible.

[0051] 4) 50nM rapamycin group: It was further divided into three groups according to different treatment methods, namely: 50nM continuous treatment group, 50nM intermittent treatment group, and 50nM treatment for three days and then discontinuation of drug group; the specific operation was the same as that of the 25nM group.

[0052] 5) 75nM rapamycin group: It was further divided into three groups according to different treatment methods, namely: 75nM continuous treatment group, 75nM intermittent treatment group, and 75nM treatment for three days and then drug withdrawal group; the specific operation was the same as that of the 25nM group.

[0053] Cell samples were collected from the above groups on the 23rd day for flow cytometry detection of erythrocyte-specific surface antigen expression and Wright-Giemsa staining. Subsequently, samples were collected every 5 days and the above tests were repeated.

[0054] The results of rapamycin treatment showed that the cell state was relatively poor in the three usage methods of the 75nM dose group, the cell morphology was irregular, and there were many dead cells; while the three doses of the drug withdrawal group after 3 days of treatment had little or no effect on cell differentiation and maturation, so the above 6 groups of rapamycin usage were excluded. The groups of repeated experiments were: blank group, solvent group, 50nM continuous treatment group, 50nM intermittent treatment group, 25nM continuous treatment group, and 25nM intermittent treatment group, a total of 6 groups. The intervention time point was the same as before, and the intervention started on the 14th day of erythroid differentiation. The intervention interval was also 3 days. On the 23rd day of induced differentiation, a small number of cells were taken for cell surface antigen detection and Wright-Giemsa staining. After that, samples were taken every 5 days to repeat the above two experiments. The experiment showed that continuous treatment with 25nM rapamycin (low dose) on the 14th day of in vitro induction can significantly improve the efficiency of enucleation.

[0055] The treatment results showed that the induction scheme was optimized in 9 experimental groups from the two aspects of the treatment method and dosage of rapamycin in the process of induced differentiation of erythroid cells, striving to achieve the best effect of promoting denucleation under the best use of rapamycin and the lowest concentration. From the treatment method, whether at the concentration of 75nM, 50nM, or 25nM, the effect of removing rapamycin after 3 days of treatment was not significantly different from that of the blank group and the solvent group; the effects of continuous treatment and intermittent treatment on promoting denucleation of erythrocytes were more obvious; therefore, continuous treatment or intermittent treatment should be selected. From the concentration of rapamycin used, the difference in the effects of low, medium, and high concentrations in the early stage was not obvious, but in the middle and late stages, the effect of 50nM and 25nM on promoting cell denucleation was obvious. At the concentration of 75nM, the cell morphology was obviously irregular, the overall cell fragments were more and the cells were mostly small, resulting in unclear boundaries between cells and fragments. Therefore, under the condition of continuous treatment with a low concentration of 25nM rapamycin, both the cell state and the denucleation rate can achieve the best effect.

[0056] 3. Optimization of rapamycin treatment time during erythrocyte differentiation In addition to optimizing the concentration of rapamycin, in order to further determine and optimize the optimal treatment time point during erythroid differentiation, cells were treated with 50 nM (intermediate dose) of rapamycin at the 1st, 8th, 11th and 14th day of induced erythroid differentiation to regulate the mTOR pathway activity of the obtained cells. Then, erythroid colony formation assay, cell growth curve drawing, BrdU staining, CFSE staining and PI staining and flow cytometry were performed to detect cell proliferation and apoptosis. The process of drug concentration optimization is the same, wherein erythroid induction differentiation fluid 1 is prepared by adding cytokines EPO, IL-3 and SCF to the basic induction differentiation fluid; erythroid induction differentiation fluid 2 is prepared by adding cytokines EPO and SCF to the basic induction differentiation fluid; erythroid induction differentiation fluid 3 is prepared by adding cytokine EPO to the basic induction differentiation fluid.

[0057] At the 1st, 8th, 11th and 14th day of the induced differentiation of erythrocytes, cells were treated with 50nM (intermediate dose) of rapamycin to observe whether it affects the proliferation and differentiation of erythrocytes. Each time point was divided into 3 groups: 50nM rapamycin treatment group, plus DMSO solvent group and blank control group. The specific operation is as follows: 1) Blank control group: This group did not receive any intervention treatment, and used erythroid induction medium to induce the directed differentiation of hematopoietic stem and progenitor cells.

[0058] 2) Solvent group: DMSO was added to the erythrocyte differentiation induction solution at 1 μL / mL (the same amount as 1000× rapamycin added to the treatment group) to eliminate the influence of the solvent on the experimental results, and other induction.

[0059] 3) 50 nM rapamycin group: At each set time point, rapamycin was added to the erythrocyte differentiation induction solution at a final concentration of 50 nM, and the rest of the operations remained unchanged.

[0060] The above groups were subjected to the following related experimental tests at different time points: ① Rapamycin was treated at the erythroid progenitor stage on day 1 of differentiation, and its effect on erythroid proliferation was observed by erythroid colony formation assay; ② Rapamycin treatment was performed on the 8th, 11th, and 14th days of differentiation. Cell samples were taken every 24 hours, and cell growth curves were drawn by cell counting to compare the effects of rapamycin treatment on cell proliferation at different time points.

[0061] ③ After rapamycin treatment at different time points on the 8th, 11th, and 14th days of differentiation, cell samples were taken every 3 days, and the changes in the expression of erythrocyte surface-specific antigens CD44, CD71, and CD235a were detected by flow cytometry to evaluate the effect of rapamycin treatment on the erythrocyte differentiation process.

[0062] ④ According to the test results of ③, the effects of rapamycin on erythrocyte proliferation at different time points were detected by CFDA-SE staining, PI staining and flow cytometry on the cells treated at the 8th and 11th days when the effects were more obvious.

[0063] ⑤ According to the detection results of ③, two time points, the 8th day and the 11th day, when red blood cell proliferation was significantly affected by rapamycin treatment, were selected for BrdU staining, and its effect on cell proliferation and cell cycle was further detected and confirmed by flow cytometry.

[0064] ⑥ Select the 8th day time point when red blood cell proliferation is affected by rapamycin treatment for BrdU staining, and use flow cytometry to detect and evaluate whether rapamycin treatment affects cell apoptosis.

[0065] The results of rapamycin treatment showed that the treatment of 50nM rapamycin at different time points of erythroid differentiation had different effects on erythroid proliferation. On the first day of differentiation, rapamycin treatment almost completely inhibited the formation of erythroid burst colonies (BFU-E) and also significantly inhibited erythroid colony-forming units (CFU-E). At the same time, the results of cell growth curves showed that rapamycin treatment significantly inhibited erythroid proliferation on the 8th day of erythroid differentiation in the proliferation period, and the inhibitory effect on proliferation was weakened on the 11th day of differentiation, while rapamycin treatment had little effect on erythroid proliferation on the 14th day. Further flow cytometry results were consistent with the cell counting results. The use of rapamycin to inhibit the mTOR pathway on the 8th and 11th days of erythroid differentiation significantly inhibited cell proliferation, and also led to cell cycle arrest in the G0 / G1 phase, and the proportion of cells in the S phase was significantly reduced. At the same time, erythroid-specific surface antigen staining and Annexin V staining showed that rapamycin treatment at different time points did not change the process of erythroid differentiation or affect cell apoptosis. Comprehensive analysis of the experimental results of this group shows that the use of rapamycin treatment starting on the 14th day of erythroid differentiation can avoid the rapid proliferation period during erythroid differentiation, which will not affect the number of erythrocytes produced, while at the same time utilizing the effect of rapamycin to promote the terminal denuclearization and maturation of erythrocytes, thereby improving the efficiency of erythrocyte maturation.

[0066] Experimental data Figure 2 (A) shows the dynamic changes of cell surface antigens CD235a and CD71 detected by flow cytometry at the 2nd, 4th, 6th, 8th, 12th, 16th, 20th, and 28th days during the erythroid differentiation stage; Figure 2 (B) in the figure is the percentage of the corresponding surface antigen. It can be seen that the erythroid differentiation surface antigen CD235a, namely the glycophorin molecule A (GPA), gradually increases in expression during the erythroid differentiation process. It is a marker for the late stage of erythroid differentiation and maturation, and is also a specific surface marker for mature erythrocytes. CD71, namely transferrin 1, is related to the proliferation and maturity of cells. It is a surface marker for the middle stage of erythroid differentiation. Its expression gradually decreases with the final maturation of erythrocytes, and is not expressed in terminal mature erythrocytes. The erythrocyte surface antigen induced by the present invention can support the erythroid differentiation of hematopoietic stem / progenitor cells.

[0067] Changes in the expression levels of erythroid differentiation-related genes GATA1, GATA2, SCL, EKLF, and erythroid-specific globin genes including α-Globin, β-Globin, γ-Globin, and ε-Globin at each time point. GATA1 is a regulatory factor for erythroid differentiation and globin expression, and GATA1 and GATA2 have a mutual regulatory effect (the expression level changes in the opposite trend). Figure 3The results of quantitative PCR showed that during the process of erythroid differentiation, GATA1 expression gradually increased while GATA2 expression gradually decreased. In the second stage of differentiation, GATA1 and SCL expression trends were consistent, both gradually increased, while GATA2 expression showed fluctuations rather than gradually decreased; EKLF expression level showed a gradual upward trend. Gene level detection showed normal.

[0068] After immunofluorescence staining, flow cytometry was used to detect and analyze the proportion of cells expressing various types of globin on the 16th day of erythroid differentiation. Figure 4 It is the percentage of cells expressing each type of globin on the 16th day of the second stage of differentiation. In previous studies, when traditional methods were used to induce human embryonic stem cells (ES) and induced pluripotent stem cells (iPS) to generate red blood cells, ES cell-derived erythroid cells mainly expressed embryonic ε-globin, less fetal γ-globin, and rarely adult β-globin. In the optimization process of the present invention, hematopoietic stem and progenitor cells derived from human umbilical cord blood were used. The red blood cells at this developmental stage should mainly express fetal γ-globin; the results showed that the induction scheme of the present invention well retained the globin expression characteristics of red blood cells induced by hematopoietic stem and progenitor cells derived from human umbilical cord blood.

[0069] Rapamycin was used at three concentrations: 25nM, 50nM, and 75nM. The treatment methods were divided into three types: continuous treatment, intermittent treatment, and drug withdrawal after three days of treatment. Together with the solvent group and the control group, there were 11 groups in total. DraQ5 nucleic acid staining was used to detect cell enucleation efficiency on the 23rd and 28th days of erythrocyte differentiation induction. The results of rapamycin treatment are shown in Figure 2. Figure 5 , Figure 6 , Figure 7 As shown: the cell state in the three usage methods of the 75nM dose group was relatively poor, the cell morphology was irregular, and there were many dead cells; while the three doses of the drug withdrawal group after 3 days of treatment had little or no effect on cell differentiation and maturation, so the above 6 groups of rapamycin usage were excluded. The groups for repeated experiments were: blank group, DMSO solvent group, 50nM continuous treatment group, 50nM intermittent treatment group, 25nM continuous treatment group, and 25nM intermittent treatment group, a total of 6 groups. The intervention time point was the same as before, and the intervention started on the 14th day of erythroid differentiation. The intervention interval was also 3 days. On the 23rd day of induced differentiation, a small number of cells were taken for cell surface antigen detection and Wright-Giemsa staining. After that, samples were taken every 5 days, and the above two experiments were repeated. The morphological and flow cytometric experimental data showed that continuous treatment with 25nM rapamycin (low dose) on the 14th day of in vitro induction could significantly improve the efficiency of enucleation, reaching 50% on the 28th day of induction. The experiment found that if the induction time was further extended to 40 days, the maximum enucleation efficiency could reach 70%. Figure 8The treatment with 50nM rapamycin on the first day of the erythrocyte differentiation induction stage had different effects on erythrocyte proliferation. Figure 8 (A) shows that rapamycin treatment almost completely inhibited the formation of erythroid burst colonies (BFU-E) on day 1 of differentiation initiation. Figure 8 (B) shows that the colony-forming unit of erythroid cells (CFU-E) was also significantly inhibited. The cell growth curves obtained by rapamycin treatment at different time points of erythroid differentiation showed that on the 8th day of erythroid differentiation in the proliferation stage, rapamycin treatment significantly inhibited the proliferation of erythrocytes, such as Fig. 9 Middle (A); Fig. 9 The results in (B) and (C) show that the inhibitory effect of rapamycin on proliferation on day 11 of differentiation was weaker than that on day 8, while rapamycin had little effect on erythrocyte proliferation on day 14. This indicates that during the in vitro induction of erythroid differentiation of human hematopoietic stem and progenitor cells, the proliferation period is mainly concentrated on days 8 to 10, after which the proliferation gradually stops and enters the terminal maturation stage, completing denucleation and organelle clearance.

[0070] Next, the expression of erythrocyte-specific surface antigens CD71 and CD235a at different time points was detected by flow cytometry to evaluate the effect of rapamycin treatment on the erythrocyte differentiation process at different time points of erythroid differentiation. Fig.10 The results showed that compared with the control group, rapamycin treatment on the 8th, 11th and 14th days of differentiation did not affect the expression of CD71 and CD235a, indicating that although rapamycin treatment affected cell proliferation, it did not affect the process of erythroid differentiation.

[0071] Subsequently, based on the cell counting data, multiple staining techniques combined with flow cytometry were further used to deeply analyze the effects of rapamycin on cell proliferation and cell cycle. The results of PI-stained flow cytometry showed that the use of rapamycin to inhibit the mTOR pathway on days 8 and 11 of erythroid differentiation led to G0 / G1 phase cell cycle arrest and a significant decrease in the proportion of S phase cells. Fig.11 In (A), the flow cytometry results of CFDA-SE staining also showed that rapamycin treatment significantly inhibited erythrocyte proliferation on days 8 and 11 of erythroid differentiation. Fig.11 (B); BrdU incorporation experiments were used to further accurately analyze the effects of rapamycin treatment on cell proliferation and cell cycle. Fig.12 The results were consistent with the previous results. Rapamycin treatment inhibited cell proliferation and caused G0 / G1 cell cycle arrest on days 8 and 11 of erythroid differentiation. Annexin V staining showed that rapamycin treatment at different time points did not affect cell apoptosis.

Claims

1. A method for efficiently inducing erythroid differentiation and maturation of human hematopoietic stem and progenitor cells without serum or feeder layer, characterized in that: It involves inducing and culturing CD34+ hematopoietic stem and progenitor cells in vitro with erythrocyte induction differentiation fluid, and then using the mTOR pathway inhibitor rapamycin to regulate the activity of the mTOR pathway during erythroid differentiation.

2. The method according to claim 1, characterized in that: The following steps are involved: (1) Resuspend CD34+ hematopoietic stem and progenitor cells in erythrocyte differentiation medium 1 and culture them in 5% O2; (2) Collect the cells obtained in step (1), centrifuge them, remove the supernatant, and resuspend them in red blood cell induction differentiation solution 2; determine whether to plate them based on the cell density; (3) Collecting the cells obtained in step (2), centrifuging them, removing the supernatant, and resuspending them in red blood cell induction differentiation solution 3; (4) adding 25-50 nM of rapamycin, an mTOR pathway inhibitor, to regulate the mTOR pathway activity of the cells obtained in step (3), and continuing the treatment for 8-14 days; Among them, erythroid induction differentiation fluid 1 is a basic induction differentiation fluid with the addition of cytokines EPO, IL-3 and SCF; erythroid induction differentiation fluid 2 is a basic induction differentiation fluid with the addition of cytokines EPO and SCF; erythroid induction differentiation fluid 3 is a basic induction differentiation fluid with the addition of cytokine EPO.

3. The method according to claim 2, characterized in that: Step (4) is to add 25 nM of mTOR pathway inhibitor rapamycin after 14 days of in vitro induction.

4. The method according to claim 3, characterized in that: Step (4) After adding the mTOR pathway inhibitor rapamycin, the basal induction differentiation medium containing the corresponding concentration of rapamycin was replaced every 2 to 3 days.

5. The method according to claim 2, characterized in that: The formula of the basic induction differentiation solution is as follows: Glutamate 4mmol / L, Penicillin 100 U / mL, Streptomycin 100 μg / mL, Inositol 4 μg / mL, Folic acid 10 μg / mL, Ferrous sulfate 0.9 μg / mL, Ferric nitrate 0.09 μg / mL, Human transferrin 150μg / mL, Recombinant human serum albumin 5%, Trypsin inhibitor 1ng / mL, Sodium selenite 6.7ng / mL, Reduced glutathione 1 μg / mL, Arachidonic acid 2ng / mL, Cholesterol 220ng / mL, Linoleic acid 10ng / mL, Linolenic acid 10 ng / mL, Oleic acid 10 ng / mL, Palmitoleic acid 10ng / mL, Heparin 5 U / mL Make up to 1L with IMDM.

6. The method according to claim 2, characterized in that: The culture time in step (1) is 7 days, and the liquid is replaced every 2 to 3 days during the culture period.

7. Red blood cells derived from human hematopoietic stem and progenitor cells obtained according to the method according to any one of claims 1 to 6.

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