CN111607566B - Method and application of differentiating human pluripotent stem cells into hematopoietic progenitor cells - Google Patents

Abstract

The invention provides a method and application for differentiating human pluripotent stem cells into hematopoietic progenitor cells. The present invention also provides hematopoietic progenitor cells differentiated from pluripotent stem cells and having a CD34+KDR+CD43-CD73-phenotype. The method of the invention can efficiently and rapidly produce hematopoietic progenitor cells with stable differentiation effect under the serum-free condition, and the hematopoietic progenitor cells are hematopoietic progenitor cells with differentiation ability of erythroid line, myeloid line and lymph line.

Description

Method and application of differentiating human pluripotent stem cells into hematopoietic progenitor cells

technical field

The present invention relates to the field of biotechnology, more specifically, to a method for differentiating human pluripotent stem cells into hematopoietic progenitor cells and applications thereof.

Background technique

Hematological diseases are diseases that originate in the hematopoietic system, or affect the hematopoietic system accompanied by abnormal blood changes, which often manifest as anemia, bleeding, fever and other symptoms.

The incidence of malignant cancer in children in my country is on the rise. According to the data as of 2014, among the malignant tumors in children, the incidence of leukemia ranks first, accounting for about one-third. For malignant hematological diseases, the clinical effect of chemotherapy is often not ideal. Since Professor Thomas first carried out hematopoietic stem cell (HSC) transplantation in the middle of the twentieth century, HSC transplantation has been widely used in the clinical treatment of leukemia, and has become a treatment for acute leukemia, malignant lymphoma, severe aplastic anemia and other diseases. one of the effective means.

Currently, HSCs are mainly derived from umbilical cord blood, bone marrow and peripheral blood. HSC transplantation is mainly divided into autologous and allogeneic HSC transplantation. Although autologous transplantation has the advantages of no graft rejection, no graft-versus-host disease and other complications, the number of autologous HSCs stored in cord blood banks is in short supply, which limits its clinical application in diseases. Although the long-term curative effect of allogeneic transplantation is better than that of autologous transplantation and the recurrence rate is low, the matching efficiency is extremely low and the source is limited, which limits the clinical application of allogeneic HSC transplantation.

Therefore, there is an urgent need in this field to find HSC resources that are safer, lower in cost and stable in source. Human pluripotent stem cells have the ability to differentiate into almost all types of somatic cells, including hematopoietic stem cells. Human pluripotent stem cells include human embryonic stem cells (ESC) and human induced pluripotent stem cells (iPSC).

Studies have shown that mouse, monkey, and human ESCs can be induced to differentiate into various blood cells in vitro, but ESCs are derived from embryos in the early stages of development, and there are problems such as difficulty in obtaining materials, immune rejection, and ethics.

Human induced pluripotent stem cells (iPSC) can be reprogrammed from human skin, blood and other somatic cells in vitro, and have similar unlimited proliferation ability as ESC, and can differentiate into almost all functional cells in vitro, including hematopoietic cells. capacity of stem cells. This characteristic of iPSC successfully circumvents the two most critical issues of immune rejection and ethics, and provides the possibility for the clinical application of hematopoietic stem cells obtained from in vitro sources for clinical transplantation.

At present, many studies have reported the methods of inducing human pluripotent stem cells to differentiate into various types of hematopoietic progenitor cells in vitro.

Chadwick K et al. reported that CD45-positive hematopoietic progenitor cells can be obtained from human pluripotent stem cells by using the embryoid body differentiation method under the effect of exogenously added BMP-4 and cytokine mixture (Chadwick K et al., 2003).

Ledran MH et al. reported a method for obtaining CD34-positive hematopoietic progenitor cells by co-culturing human pluripotent stem cells with mouse-derived stromal cells (or trophoblast cells) (Ledran MH et al., 2008).

Yu C et al. reported the method of inducing the differentiation of human pluripotent stem cells into CD34 and CD45 positive hematopoietic progenitor cells in a chemically defined differentiation medium (Yu C et al., 2010).

However, the hematopoietic progenitor cells obtained by the above method only have the ability to differentiate into erythroid and myeloid blood cells, but not the ability to differentiate into lymphoid blood cells (i.e. T cells, B cells and NK cells), so they do not have the ability to rebuild the entire The capacity of the hematopoietic system.

On this basis, Kennedy M et al obtained mature hematopoietic progenitor cells with T cell differentiation ability under the condition of serum-free and stromal-free cells, and proved that the acquisition of mature hematopoietic progenitor cells requires the combination of surface markers CD34+CD43 - at the hemogenic endothelium (HE) stage, and its function can be evaluated by T cell differentiation ability (Kennedy M et al., 2013).

Uenishi GI et al. found that the activation of the Notch signaling pathway is crucial in the formation of mature HE (Uenishi GI et al., 2015).

Although the process of in vitro differentiation of human pluripotent stem cells into hematopoietic progenitor cells has been understood in the art, existing differentiation methods have some disadvantages, including unstable differentiation effect, low efficiency, or the use of serum-containing Culture system or trophoblast cells, so it is not suitable for the subsequent production of clinical-grade cell preparations.

Therefore, there is an urgent need in this field to develop a method for efficiently and rapidly preparing differentiated and stable hematopoietic progenitor cells under serum-free conditions.

Contents of the invention

The purpose of the present invention is to provide an efficient, fast, serum-free method for preparing differentiated and stable hematopoietic progenitor cells

In the first aspect of the present invention, a hematopoietic progenitor cell is provided, the hematopoietic progenitor cell is formed by differentiation of pluripotent stem cells, and has a CD34+KDR+CD43-CD73- phenotype.

In another preferred embodiment, the pluripotent stem cells are selected from the group consisting of embryonic stem cells (ESC), induced pluripotent stem cells (iPSC) or a combination thereof.

In another preferred example, the hematopoietic progenitor cells have a CD34+KDR+CD43-CD73-DLL4+ phenotype.

In another preferred example, the hematopoietic progenitor cells have a CD34+KDR+CD43-CD73-DLL4+CD184- phenotype.

In another preferred example, the hematopoietic progenitor cells are human hematopoietic progenitor cells.

In another preferred embodiment, the pluripotent stem cells are human pluripotent stem cells, including human embryonic stem cells (ESC) and human induced pluripotent stem cells (iPSC).

In another preferred example, the hematopoietic progenitor cells are a population of hematopoietic progenitor cells.

In another preferred example, the hematopoietic progenitor cells have any one or more characteristics selected from the following group (A):

(i) more than 90% of the cells have the surface antigen CD34;

(ii) More than 90% of the cells have the surface antigen combination CD34+KDR+;

(iii) more than 90% of the cells have the surface antigen combination CD34+CD43-;

(iv) More than 90% of the cells have the surface antigen combination CD34+CD73-;

(v) more than 80% of the cells have the surface antigen combination CD34+DLL4+; and

(vi) More than 70% of cells have surface antigen combination CD34+CD184-.

In another preferred example, the hematopoietic progenitor cells have 3, 4, 5 or more than 6 characteristics of group (A).

In another preferred example, the hematopoietic progenitor cells have the ability to differentiate into CD43+CD45+ blood precursor cells.

In another preferred example, the hematopoietic progenitor cells have the ability to differentiate into erythroid blood cells.

In another preferred example, the hematopoietic progenitor cells have the ability to differentiate into myeloid blood cells.

In another preferred example, the hematopoietic progenitor cells also have the ability to differentiate into lymphocytes.

In the second aspect of the present invention, there is provided a pharmaceutical composition for treating hematological diseases, said pharmaceutical composition comprising: an effective amount of the hematopoietic progenitor cell described in the first aspect of the present invention, and a pharmaceutically acceptable The carrier that accepts .

In another preferred example, the pharmaceutical composition is a liquid preparation.

In another preferred example, the pharmaceutical composition is a cell preparation.

In another preferred example, the pharmaceutical composition is an intravenous injection reagent.

In another preferred example, the pharmaceutically acceptable carrier includes (but is not limited to): saline, buffer, glucose, water, DMSO, and combinations thereof.

In another preferred example, the concentration of the hematopoietic progenitor cells is 1×10 ³ cells/ml-1×10 ⁷ cells/ml, preferably 1×10 ⁴ -1×10 ⁶ cells/ml, more preferably The ground is 1×10 ⁵ /ml-9.9×10 ⁵ /ml.

In another preferred example, the blood disease is selected from the group consisting of anemia, leukemia, or a combination thereof.

In a third aspect of the present invention, a serum-free method for preparing hematopoietic progenitor cells is provided, comprising the steps of:

(a) providing pluripotent stem cells;

(b) performing suspension culture on the pluripotent stem cells to form an embryoid body (embryoid body, EB);

(c) in the presence of a compound GSK-3β inhibitor, the embryoid body is induced and cultured to form a mesoderm;

(d) in the presence of a compound TGF-β inhibitor, the mesoderm is induced and cultured to form hematopoietic endothelial cells;

(e) transforming and culturing the hematopoietic endothelial cells in the presence of a combination of blood cell growth factors, so as to obtain the hematopoietic progenitor cells described in the first aspect of the present invention.

In another preferred embodiment, step (a) includes: digesting the pluripotent stem cells to form a single cell suspension.

In another preferred embodiment, it is treated with accutase digestive enzyme.

In another preferred example, in step (b), the seeding concentration of pluripotent stem cells is 0.1×10 ⁶ -5×10 ⁶ /ml.

In another preferred embodiment, in step (b), culture is carried out in CD34A or pluripotent stem cell maintenance medium added with ROCK inhibitor or other compounds that promote the survival of single human pluripotent cells.

In another preferred embodiment, ROCK inhibitors or other compounds that promote the survival of single human pluripotent cells are selected from the group consisting of Blebbistatin, HA-100, Y-27632, HA-1077, KD-025, Y-33075, Narciclasine or its combination.

In another preferred example, in step (b), the ROCK inhibitor is Blebbistatin.

In another preference, in step (b), the time of suspension culture is 4-24 hours, 8-24 hours, 12-24 hours, 16-24 hours, 4-32 hours, 8-32 hours, 12 hours -32 hours, 16-32 hours, or 12-24 hours.

In another preferred example, in step (c), the GSK-3β inhibitor can be one of the following compounds or a combination thereof: NP031112, TWS119, SB216763, CHIR-98014, AZD2858, AZD1080, SB415286, LY2090314 and CHIR99021.

In another preferred example, in step (c), the GSK-3β inhibitor may be CHIR99021.

In another preferred example, in step (c), the concentration of the GSK-3β inhibitor can be 0.1-20uM, 0.5-20uM, 1-20uM or 2-20uM.

In another preference, in step (c), the culture time is 1-3.5 days, 1.5-3.5 days, 2-3.5 days, 1-3 days, 1.5-3 days, or 2-3 days.

In another preferred example, in step (c), BMP-4, bFGF and VEGF also exist in the culture system.

In another preference, in step (c), the concentration of BMP-4 is 0-50ng/mL; the concentration of bFGF is 0-50ng/mL; and the concentration of VEGF is 1-100ng/mL.

In another preference, in step (d), the TGF-β inhibitor can be one of the following compounds or a combination thereof: A-83-01, GW6604, IN-1130, Ki26894, LY2157299, LY364947 ( HTS-466284), LY550410, LY5 73636, LY580276, NPC-30345, SB-431542, SB-505124, SD-093, Sm16, SM305, SX-007, Antp-Sm2A, LY2109761.

In another preferred example, in step (d), the TGF-β inhibitor may be SB431542.

In another preferred example, in step (d), the concentration of the TGF-β inhibitor can be 0.1-10uM, 0.3-10uM, 0.5-10uM, or 1-10uM.

In another preference, in step (d), the culture time can be 0.5-1 day, 0.5-2 day, 0.5-3 day, 0.5-3.5 day, 0.5-4 day, or 1-2 day.

In another preferred example, in step (d), BMP-4, bFGF and VEGF also exist in the culture system.

In another preferred example, in step (d), the concentration of BMP-4 is 0-50 ng/mL; the concentration of bFGF is 0-20 ng/mL; and the concentration of VEGF is 1-100 ng/mL.

In another preferred example, in step (e), bFGF and VEGF also exist in the culture system.

In another preferred example, in step (e), the culture system does not contain BMP-4.

In another preference, in step (e), the blood cell growth factor combination includes SCF and TPO.

In another preferred example, the combination of blood cell growth factors further includes one or more blood growth factors selected from the following group: FLT3L, IL3, IL6, IGF1, IL11, IL7, IL15, or a combination thereof.

In another preferred example, the combination of blood cell growth factors is selected from the following group:

1) SCF, TPO;

2) SCF, TPO, FLT3L;

3) SCF, TPO, FLT3L, IL3, IL6;

4) SCF, TPO, FLT3L, IL3, IL6, IGF1;

5) SCF, TPO, FLT3L, IL3, IL6, IGF1, IL11;

6) SCF, TPO, FLT3L, IL3, IL6, IGF1, IL11, IL7; and

7) SCF, TPO, FLT3L, IL3, IL6, IGF1, IL11, IL7, IL15.

In another preferred example, the concentration of each of the blood cell growth factors is as follows:

The concentration of SCF is 10-500ng/mL, and the preferred concentration can be 100ng/mL;

TPO concentration is 10-300ng/mL, preferably 100ng/mL;

The concentration of FLT3L is 5-300ng/mL, preferably 50ng/mL;

The concentration of IL3 is 5-300ng/mL, preferably 50ng/mL;

The concentration of IL6 is 5-300ng/mL, preferably 50ng/mL;

IGF1 concentration is 10-100ng/mL, preferably 25ng/mL;

The concentration of IL11 is 5-200ng/mL, preferably 10ng/mL;

The concentration of IL7 is 1-200ng/mL, and the preferred concentration can be 10ng/mL;

The concentration of IL15 is 5-200ng/mL, preferably 20ng/mL.

In another preferred example, in steps (c), (d) and/(e), the culture is suspension culture.

In another preferred example, the method also includes:

(f) Examining the cell phenotype of the formed hematopoietic progenitor cells.

In another preferred example, the method is a method for preparing hematopoietic progenitor cells under serum-free conditions.

In the fourth aspect of the present invention, the use of the hematopoietic progenitor cells described in the first aspect of the present invention is provided for the preparation of drugs for the treatment of blood diseases.

In another preferred example, the drug is a liquid preparation.

In another preferred example, the blood disease is selected from the group consisting of anemia, leukemia, or a combination thereof.

In the fifth aspect of the present invention, there is provided a method for treating hematological diseases, comprising the steps of: administering the hematopoietic progenitor cells described in the first aspect of the present invention to a subject in need, or administering a pharmaceutical composition containing hematopoietic progenitor cells.

In another preferred example, the subject is a human or a non-human mammal, preferably a human.

In another preferred example, the site of administration is within the subject's vein or bone marrow.

It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described in the following (such as embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, I will not repeat them here.

Description of drawings

Figure 1 shows a flow chart of one embodiment of the present invention.

Figure 2 shows embryoid bodies formed from iPSCs in one embodiment of the present invention.

Figure 3 shows differentiation from embryoid body to mesoderm in one embodiment of the present invention.

Fig. 4 shows the differentiation of mesoderm cells into hematopoietic endothelium (HE) in one embodiment of the present invention.

Figure 5 (5A-5D) shows the identification results of cell phenotypes in one embodiment of the present invention.

Figure 6 (6A-6D) shows the results of identification of cell phenotypes after enrichment of hematopoietic progenitor cells in one embodiment of the present invention.

Figure 7 (7A-7B) shows the further refined identification results of hematopoietic progenitor cell phenotypes in one embodiment of the present invention.

Fig. 8 (8A-8C) shows the comparison experiment between the technology optimization process of the present invention and the known method. Compared with the known method without CHIR99021 (CHIR99021 concentration is 0), CHIR99021 treatment significantly improved the differentiation efficiency of pluripotent stem cells to hematopoietic progenitor cells, the data expressed as the proportion of CD34+KDR+ cells in EBs significantly increased after CHIR99021 treatment Improvement, reaching the optimum when the concentration of CHIR99021 is 6uM.

Fig. 9 shows another comparison experiment between the optimization process of the technology of the present invention and the known method. Compared with the method without SB431542 (SB431542 treatment time was 0 hours), SB431542 treatment significantly improved the differentiation efficiency of pluripotent stem cells to hematopoietic progenitor cells, and the data were expressed as the proportion of CD34+CD43- cells in EBs after SB431542 treatment. Significantly improved, the processing time of SB431542 is 24 hours to reach the optimum.

Figure 10 shows that the concentration of bFGF in the method is optimized during the technical optimization process of the present invention. Data show the effect of bFGF on the proportion of CD34+KDR+ cells in EBs.

Fig. 11 shows another comparative experiment in the optimization process of the technology of the present invention and another method. The data showed that the combined treatment of CHIR99021 and SB431542 significantly increased the proportion of KDR+CD73- cells in the CD34+CD43- population compared with no treatment of CHIR99021 and SB431542, making the phenotype of differentiated hematopoietic progenitor cells more uniform. In the figure, CHIR indicates CHIR99021, and SB indicates SB431542.

Figure 12 shows that CD34+CD43-KDR+CD73- cells can be efficiently differentiated into CD43+CD45+ blood precursor cells. In the figure, CHIR indicates CHIR99021, and SB indicates SB431542.

Figure 13 shows that KDR+CD34+CD43-CD73- cells have the ability to differentiate into various blood cells.

Detailed ways

After extensive and in-depth research, the present inventors unexpectedly found for the first time that in the presence of a specific compound, hematopoietic progenitor cells with stable differentiation effects can be efficiently and rapidly produced under serum-free conditions through optimization of conditions, and said Hematopoietic progenitor cells are hematopoietic progenitor cells that have the ability to differentiate into erythroid, myeloid, and lymphoid lines. The present invention has been accomplished on this basis.

the term

As used herein, the terms "above" and "below" are inclusive, such as "above 95%" means > 95%, and "below 0.2%" means < 0.2%.

The term "pluripotent" refers to stem cells that have the potential to differentiate into all cells of one or more tissues or organs, e.g., any of the three germ layers; endoderm (inner stomach lining, gastrointestinal tract, lungs) , mesoderm (muscle, bone, blood, genitourinary) or ectoderm (epidermal tissue and nervous system).

"Pluripotent stem cells" refers to cells capable of giving rise to cells of all three germinal layers, namely endoderm, mesoderm and ectoderm. Although theoretically pluripotent stem cells can differentiate into any cell in the body, experimental assays of pluripotency are often based on the differentiation of pluripotent cells into several cell types per germinal layer.

The term "induced pluripotent stem cells", often abbreviated as iPS cells or iPSCs, refers to the transformation of non-pluripotent cells (usually adult somatic cells) or terminally differentiated cells (such as fibroblasts) into Cells, hematopoietic cells) artificially prepared pluripotent stem cells, such as muscle cells, neurons, epidermal cells, etc.

The term "embryonic stem cells", often abbreviated ES, are pluripotent stem cells derived from early embryos.

The term "suspension culture" refers to a culture in which cells or cell aggregates propagate while suspended in a liquid medium.

The term "differentiation" is the process by which less specialized cells form the progeny of at least one new, more specialized cell type.

The term "embryoid body", ie embryoid body or aggregate, refers to a homogeneous or heterogeneous cluster of cells comprising differentiated cells, partially differentiated cells and/or pluripotent stem cells in suspension culture. Certain aspects of the invention may use three-dimensional embryoid bodies as an intermediate step in order to recapitulate some of the cues inherent to in vivo differentiation. At the onset of cell aggregation, differentiation can be initiated and cells can initiate to a limited extent to recapitulate embryonic development. Although they cannot form trophectoderm tissue, virtually every other type of cell present in the organism can develop. The present invention can further promote the differentiation of hematopoietic progenitor cells after embryoid body formation.

As used herein, the term "blood disease" refers to diseases related to cell lesions in the blood. Representative blood diseases include (but are not limited to): anemia, thrombocytopenia, leukemia, lymphoma, severe aplastic anemia, multiple Myeloma.

As used herein, the term "CHIR99021" refers to the compound whose CAS No. is 252917-06-9. It should be understood that the term also includes CHIR99021 and its salts, especially pharmaceutically acceptable salts. The chemical structure of CHIR99021 is:

As used herein, the term "SB431542" refers to the compound with CAS No. 301836-41-9. It should be understood that the term also includes SB431542 and salts thereof, especially pharmaceutically acceptable salts. The specific chemical structure is:

Preparation

The present invention provides a method for rapidly and efficiently inducing differentiation of human pluripotent stem cells into hematopoietic progenitor cells under specific serum-free conditions based on specific small molecule compounds.

The main features of the method of the present invention include: (1) using the small molecular compound CHIR99021 to induce mesoderm, thereby obtaining a rapid and uniform mesoderm; (2) using SB431542 to induce hematopoietic endothelium (HE), thereby removing primitive hematopoietic cells (3) The cell phenotype of the final product prepared by the method of the present invention is CD34+KDR+CD43-CD73-, which is a mature hematopoietic progenitor cell with efficient T cell differentiation ability.

In the present invention, in addition to using specific small molecule compounds CHIR99021 and SB431542 at different culture stages, conditions such as the concentration and culture time of the small molecule compounds are also optimized, so that the optimized method of the present invention can not only quickly , Efficiently prepare hematopoietic progenitor cells, and the prepared hematopoietic progenitor cells have the ability to stably differentiate into a variety of different blood cells (including erythroid, myeloid and lymphoid cells at the same time).

It should be understood that in the method of the present invention, in addition to using specific small molecule compounds CHIR99021 and SB431542 at different culture stages, medium and culture components known in the art can be used. In order to meet the requirements of clinical-grade cell preparations, the culture medium or culture system of the present invention is preferably serum-free. In addition, in the medium or culture system, other components that are beneficial to induce differentiation can also be added, such as other small molecule compounds, including additional GSK3beta inhibitors and TGFbeta inhibitors.

In the present invention, representative additional GSK3beta inhibitors include: BIO (CAS No. 667463-62-9), TWS119 (CAS No. 601514-19-6), Kenpaullone (CAS No. 142273-20-9) , and Indirubin-3'-oxime (CAS No.160807-49-8), etc.

In the present invention, representative additional TGFbeta inhibitors include: RepSox (CAS No. 446859-33-2), A8301 (CAS No. 909910-43-6), SD208 (CAS No. 627536-09-8) wait.

hematopoietic progenitor cells

As used herein, the term "hematopoietic progenitor cell of the present invention" refers to the hematopoietic progenitor cell formed by directed differentiation from pluripotent stem cells, as described in the first aspect of the present invention. Unless otherwise stated, "hematopoietic progenitor cells" refers to cells characterized by a CD34+KDR+CD43-CD73- phenotype. A particularly preferred hematopoietic progenitor cell is a CD34+KDR+CD43-CD73-purified cell population.

The hematopoietic progenitor cell of the present invention is a hematopoietic progenitor cell with differentiation ability of erythroid line, myeloid line and lymphoid line.

The invention also provides cell preparations comprising the hematopoietic progenitor cells of the invention. These cell preparations can be used to treat blood diseases such as leukemia.

Those of ordinary skill in the art can use, process, administer and other operations on hematopoietic progenitor cells using conventional methods. For example, each batch of hematopoietic progenitor cells must pass sterility, endotoxin, and mycoplasma inspections, as well as DNA identity verification, before distribution or use. Each batch of released cells must comply with cell viability ≥ 95% and cell purity (positive indicators ≥ 95%, negative indicators < 2%). Hematopoietic progenitor cell acute toxicity and allergy test results were negative.

Antigen detection of hematopoietic progenitor cells

The hematopoietic progenitor cells prepared by the method of the present invention can be verified by detecting cell surface antigens.

CD34 antigen is a highly glycosylated single transmembrane protein, which is selectively expressed on the surface of human hematopoietic stem cells (HSC), hematopoietic progenitor cells (HPC) and vascular endothelial cells (EC). In the present invention, after purification and enrichment, the proportion of hematopoietic progenitor cells with CD34 in the total cell population is preferably ≥ 90%.

The KDR antigen is a vascular endothelial growth factor (VEGF) receptor that is widely expressed in a variety of mesoderm tissues during development and expressed in vascular endothelial cells at the embryonic stage. In vitro and in vivo data show that KDR is essential for the development and formation of vascular endothelial cells and hematopoietic cells. In the present invention, after purification and enrichment, the proportion of hematopoietic progenitor cells with KDR in the total cell population is preferably ≥ 90%.

CD43 antigen is a glycoprotein encoded by the SNP gene, also known as leukocyte sialoglycoprotein or sialic acid protein, expressed on the surface of most blood leukocytes such as B cells, T cells, NK cells, and granulocytes. In the present invention, the CD43 negative characteristic is used to mark the hematopoietic progenitor cells differentiated from iPSCs. In the present invention, after purification and enrichment, the proportion of CD43-negative hematopoietic progenitor cells in the total cell population is preferably ≥ 90%.

CD73 antigen is an extracellular-5' nucleotidase and a signal transduction molecule expressed on some subsets of T cells and B cells, epithelial cells, endothelial cells and mesenchymal stem cells. In the present invention, the CD73-negative characteristic is used to mark hematopoietic progenitor cells differentiated from pluripotent stem cells (such as iPSCs). In the present invention, after purification and enrichment, the proportion of CD73-negative hematopoietic progenitor cells in the total cell population is preferably ≥ 90%.

DLL4 refers to Delta-like ligand 4, which is one of the ligands of the Notch signaling system family in mammals, and is specifically expressed on the cell surface of the vascular endothelial system. Studies have shown that DLL4 does cause developmental disorders of the vasculature. Studies have also shown that the Notch signaling pathway plays an important role in the differentiation of hematopoietic progenitor cells into lymphocytes. In the present invention, the DLL4 expressed on the surface of the cell membrane is used as one of the characteristics to characterize the phenotype of hematopoietic progenitor cells. In the present invention, after purification and enrichment, the proportion of DLL4-positive hematopoietic progenitor cells in the total cell population is preferably ≥ 80%.

CD184, also known as CXCR4, is a G protein-coupled chemokine receptor with 7 transmembrane structures. CD184 is mainly expressed on the surface of resting T cells. In the present invention, CD184 negative (no expression) is used as one of the characteristics of the phenotype of hematopoietic progenitor cells. In the present invention, after purification and enrichment, the proportion of CD184-negative hematopoietic progenitor cells in the total cell population is preferably ≥ 70%.

The purity and degree of differentiation of the hematopoietic progenitor cells of the present invention can be detected by general methods, such as flow cytometry. During detection, different specific antibodies targeted to corresponding cell surface antigens are added, and the antibody can be a complete monoclonal or polyclonal antibody, or an antibody fragment with immunological activity, such as Fab' or (Fab) ₂ fragments; single chain Fv molecules (scFV); or chimeric antibodies. Antibodies are added to bind to antigens on the cell surface for a certain period of time, and cells can be automatically analyzed and/or sorted by flow cytometry.

Pharmaceutical composition and its application

The present invention also provides a pharmaceutical composition, which contains an effective amount of the hematopoietic progenitor cells induced and differentiated from pluripotent stem cells of the present invention, and a pharmaceutically acceptable carrier.

Usually, the hematopoietic progenitor cells of the present invention can be prepared in a non-toxic, inert and pharmaceutically acceptable aqueous carrier medium, such as physiological saline, wherein the pH is usually about 6-8, preferably, the pH is about It is 6.5-7.5.

As used herein, the term "effective amount" or "effective dose" refers to the amount that can produce functions or activities on humans and/or animals and can be accepted by humans and/or animals.

As used herein, a "pharmaceutically acceptable" ingredient is one that is suitable for use in humans and/or mammals without undue adverse side effects (such as toxicity, irritation, and allergic reactions), i.e., that has a reasonable benefit/risk ratio . The term "pharmaceutically acceptable carrier" refers to a carrier for the administration of a therapeutic agent, including various excipients and diluents.

The carrier contained in the pharmaceutical composition of the present invention includes (but is not limited to): saline, buffer solution, glucose, water, glycerol, ethanol, and combinations thereof. Generally, the pharmaceutical preparation should match the mode of administration, and the pharmaceutical composition of the present invention can be made into an injection form, for example, using normal saline or an aqueous solution containing glucose and other adjuvants to prepare by conventional methods. The pharmaceutical composition is preferably produced under sterile conditions. The amount of active ingredient administered is a therapeutically effective amount. The pharmaceutical preparations of the present invention can also be made into sustained-release preparations.

The effective amount of the hematopoietic progenitor cells of the present invention may vary with the mode of administration and the severity of the disease to be treated. The selection of a preferred effective amount can be determined by those of ordinary skill in the art based on various factors (e.g., through clinical trials). The factors include but are not limited to: the pharmacokinetic parameters such as bioavailability, metabolism, half-life, etc.; the severity of the disease to be treated by the patient, the body weight of the patient, the immune status of the patient, the route of administration, etc. .

The pharmaceutical composition of the present invention is preferably an intravenous injection reagent. In another preferred example, in the intravenous injection reagent, the concentration of the hematopoietic progenitor cells is 1×10 ³ cells/ml-1×10 ⁷ cells/ml, preferably 1×10 ^4-1 ×10 ⁶ cells/ml, more preferably 1×10 ⁵ cells/ml-9.9× ^{10 5} cells/ml.

The present invention also provides a method of using the pharmaceutical composition of the present invention. Typically, the method comprises the step of administering hematopoietic progenitor cells to a subject in need thereof.

In the present invention, hematopoietic progenitor cells are administered to the subject in need, preferably in the subject's vein, so as to treat the corresponding blood disease.

In the present invention, representative blood diseases include (but are not limited to): anemia, leukemia, thrombocytopenia, lymphoma, severe aplastic anemia, multiple myeloma.

The main advantages of the present invention include:

First, the hematopoietic progenitor cells obtained by the method have the ability to differentiate to lymphoid blood cells including T cells, and have great potential for clinical application;

Second, by using small molecular compounds to fine-tune the differentiation process, stable and efficient differentiation is achieved, and the proportion of CD34-positive hematopoietic progenitor cells in the final culture system can reach more than 30%; CD34+KDR+CD43-CD73-expression Type cells account for more than 80% of CD34+ cells, and have a strong ability to differentiate into T cells;

Third, the entire differentiation process uses serum-free medium and does not use trophoblast cells, which is suitable for the production of subsequent clinical-grade cell preparations;

Fourth, the whole process of this method adopts the method of embryoid body suspension shaking culture, which is suitable for the production of large-scale cell preparations.

Below in conjunction with specific embodiment, further illustrate the present invention. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. Experimental methods not indicating specific conditions in the following examples are usually according to conventional conditions, such as Sambrook et al., Molecular cloning: the conditions described in the laboratory manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the manufacturer's suggested conditions. Percentages and parts are by weight unless otherwise indicated.

Material

The composition of CD34A medium is as follows:

serial number Element Final concentration 1 Transferrin 2-200μg/ml 2 MTG 100-400μM 3 Vitamin C 20-100μg/ml 4 Glutamine 0.5-5mM 5 recombinant human insulin 0.5-5ug/mL 6 basal medium the

Preferably. The medium of the present invention is a serum-free medium with clear chemical composition. In addition, in the basal medium, components such as transferrin, recombinant human insulin, 1-thioglycerol, vitamin C, and glutamine can be added.

Transferrin (transferrin), also known as transferrin, is the main iron-containing protein in plasma, responsible for transporting iron absorbed by the digestive tract and released by the degradation of red blood cells. The transferrin mentioned in the present invention refers to recombinant human transferrin, which does not contain animal-derived components, and has the same biological activity as the natural protein.

MTG refers to the organic compound 1-thioglycerol (1-Thioglycerol), CAS No. 96-27-5, and the molecular formula is HSCH2CH(OH)CH2OH.

Vitamin C includes vitamin C or its various forms of salts or its various forms of derivatives.

Glutamine refers to L-glutamine (L-Glutamine), is the coded amino acid in the protein synthesis, non-essential amino acid of mammal, and is used as the necessary supplement of cell culture in the present invention.

Recombinant human insulin refers to the recombinant human insulin protein produced by recombinant DNA technology, which has the same biological activity as natural human insulin protein.

The basal medium belongs to the chemically defined medium, such as Iscove's modified Dulbecco'smedium (IMDM liquid medium), Eagle's Basal Medium (BME), Eagle MEM, DMEM, Ham, RPMI1640 and Fischer medium and other basal cell culture medium, Variations or combinations thereof.

Example 1

Embryoid body (EB) formation

In this example, the well-undifferentiated iPSCs cultured to a degree of polymerization of 90% were digested into a single-cell suspension, resuspended in human pluripotent stem cell culture medium at a certain density, and placed in a 37°C incubator Shake and culture overnight on a shaker to form embryoid bodies (embryoid bodies, EBs) that are relatively uniform in size and shape. The experimental method is as follows:

1.1. Culture of human iPSCs

The human iPSCs used in this example have undergone strict pluripotency verification (expressing various pluripotency markers, and can form teratomas including inner, middle and outer germ layers in immunodeficient mice). iPSCs are normally cultured in iPSC maintenance medium, the medium used is E8 or TeSR or other similar medium.

1.2 Formation of EB

The embryoid body formation experiment was performed when iPSCs were cultured to 90% degree of polymerization according to the above method. The specific operation is: using mechanical or enzymatic methods known in the art to dissociate the pluripotent stem cells into substantially individual cells, such as accutase digestion enzyme to digest iPSCs into a complete single-cell suspension, and dissociate the dispersed pluripotent cells Seed in CD34A or pluripotent stem cell maintenance medium at a density of about 0.1 million to about 5 million/mL. In some aspects, ROCK inhibitors or other small molecular compounds that can effectively improve cloning efficiency and cell survival can be added to the medium, such as Blebbistatin, HA-100, Y-27632, HA-1077, KD-025, Y-33075, Narciclasine, or combinations thereof, may be used in effective concentrations. For example, at least or about 2.5, 3.0, 4.5, 5.5, 6.0, 7.0, 8.0, 9.0, 10.0, 11.0, 12.0, to about 12.5 uM, or any concentration range therein. Place this cell suspension in a 37°C incubator to culture in a non-static manner such as shaking, rotating or stirring, such as a large-capacity bioreactor, any culture that keeps the cells at a controlled moving speed. Agitation improves the circulation of nutrients and cellular waste, and also provides a more uniform environment to control cell aggregation. For example, the rotational speed can be set to about 6, 15, 30, 40, 50, 60, 70, 80, 90, 100rpm, or any range therein; the incubation period of non-static cultivation in this step can be about 4-24 hours, 8-24 hours, 12-24 hours, 16-24 hours, 4-32 hours, 8-32 hours, 12-32 hours, 16-32 hours, 12-24 hours or any range derivable therein. At the end of the culture, EBs with relatively uniform size and shape are obtained, and the diameter of the cell embryoid body can be about 50, 65, 75, 85, 95, 105, 115, 125, 135, 145, 155, 170, 185, 200uM , or any range of diameters therein. Diameter can be mean diameter, median or mean diameter. In another aspect, EB is at least about 25%, 35%, 45%, 55%, 70%, 85%, 95%, 99%, or any proportion thereof may comprise at least or about 10, 55, 95, 135, 175, 225, 550, 750, 1000 cells or any range derivable therein.

In the present embodiment, a T25 culture flask was used, and the concentration of Blebbistatin was 2.5uM; the culture time was 10 hours. At the end of the culture, the diameter of the EB was about 100uM (Fig. 2).

Example 2

Differentiation of embryoid bodies to mesoderm

In this example, the above-mentioned EBs of D1 were resuspended in fresh CD34A medium supplemented with BMP-4, bFGF and VEGF, and an optimized concentration of GSK3β inhibitor CHIR99021 was added to the medium, and continued in a 37°C incubator. Shaking and culturing on the internal shaker for 2 days, the EB volume continued to increase, and a more uniform mesoderm differentiation was obtained. The experimental method is as follows:

Take out the culture bottle (Example 1) that D1EB is housed from the incubator, tilt the culture bottle to make the EB sink to the bottom, remove the supernatant, wash once with IMDM basal medium, then add fresh CD34A medium, and add cells Factors BMP-4, bFGF, VEGF, and the small molecule compound CHIR99021.

In this embodiment, T25 culture flasks were used for cultivation, wherein the concentration of BMP-4 was 1 ng/mL; the concentration of bFGF was 0.5 ng/mL; the concentration of VEGF was 5 ng/mL. The concentration of CHIR99021 can be 4uM. At the end of the culture, the EB is translucent and nearly spherical, with a diameter between 150um-200um (Figure 3).

Example 3

Induce mesoderm cells to differentiate into hematopoietic endothelium (HE)

In this example, the third-day (D3) EBs prepared in Example 2 were resuspended in fresh CD34A medium supplemented with BMP-4, bFGF and VEGF, and an optimized concentration of TGFβ inhibitor was added to the medium SB431542, continue to culture on a shaker in a 37°C incubator for 1 day, the volume of EBs continues to increase, and mesoderm cells are induced to differentiate into HE cells.

The experimental method is as follows: Take out the culture flask containing D3 EB from the incubator, tilt the culture flask to make the EB sink to the bottom, remove the supernatant, wash once with IMDM basal medium, then add fresh CD34A medium, and add cells Factors BMP-4, bFGF, VEGF, and small molecule compound SB431542.

In this embodiment, T25 culture flasks were used for cultivation, wherein the concentration of BMP-4 was 0.5 ng/mL; the concentration of bFGF was 1 ng/mL; the concentration of VEGF was 5 ng/mL. The concentration of SB431542 was 6uM. Processing time is 24 hours. At the end of the culture, EBs were translucent and nearly spherical, with a diameter between 150um and 300um (Figure 4).

Example 4

Induce endothelial to hematopoietic transition (EHT)

In this example, the EBs prepared in Example 3 on day 4 (D4) were resuspended in fresh CD34A medium supplemented with VEGF and bFGF, and blood cell growth factors (including but not limited to Not limited to the combination of SCF, TPO, FLT3L, IL3, IL6, IGF1, IL11), continue to culture on a shaker in a 37°C incubator for 1-6 days to induce the occurrence of EHT process and obtain CD34-positive hematopoietic progenitor cells.

The experimental method is as follows:

Take out the culture bottle containing D4EB from the incubator, tilt the culture bottle to make the EB sink to the bottom, remove the supernatant, wash once with IMDM basal medium, then add fresh CD34A medium, and add cytokines bFGF, VEGF and blood Cell Growth Factor Combination. In this embodiment, the concentration of bFGF is 2ng/mL; the concentration of VEGF is 5ng/mL. There can be many combinations of blood cell growth factors, such as:

1) SCF 100ng/ml, TPO 50ng/ml;

2) SCF 100ng/ml, TPO 100ng/ml, FLT3L 10ng/ml;

3) SCF 100ng/ml, TPO 100ng/ml, FLT3L 10ng/ml, IL6 10ng/ml;

4) SCF 100ng/ml, TPO 100ng/ml, FLT3L 10ng/ml, IL6 10ng/ml, IGF1 25ng/ml;

5) SCF 100ng/ml, TPO 100ng/ml, FLT3L 10ng/ml, IL6 10ng/ml, IGF1 25ng/ml, IL11 10ng/ml;

6) SCF 100ng/ml, TPO 100ng/ml, FLT3L 10ng/ml, IL6 10ng/ml, IGF1 25ng/ml, IL11 10ng/ml, IL7 5ng/ml;

7) SCF 100ng/ml, TPO 100ng/ml, FLT3L 10ng/ml, IL6 10ng/ml, IGF1 25ng/ml, IL11 10ng/ml, IL7 5ng/ml, IL15 20ng/ml.

Example 5

Detection of cell phenotype

In this example, for the cells prepared in Example 4, antibodies against CD34, CD43, CD73 and KDR were used to detect the cell phenotype by flow cytometry.

In this example, all cells on the fifth day of differentiation were detected, including non-blood cells, non-hematopoietic vascular endothelial precursor cells, hematopoietic progenitor cells, and the like.

The results showed that the phenotype of the obtained cells was: CD34+CD43-KDR+CD73-; among them, the ratio of CD34+ cells to the total number of cells reached 33.21% (Fig. 5A); the ratio of CD43- cells to CD34+ cells reached 91.75% (Fig. 5B), so the ratio of CD34+CD43- cells to the total number of cells is 33.21% X 91.75% = 30.47%; the ratio of KDR+ cells to CD34+CD43- cells is 97.13% (Fig. 5C), so CD34+CD43-KDR+ cells The proportion of the total number of cells is 30.47% X 97.13% = 29.60%; the proportion of CD73- cells to CD34+CD43-KDR+ cells is 88.21% (Figure 5D), so CD34+CD43-KDR+CD73- cells accounted for the total cells The proportion of the number is 29.60% X 88.21% = 26.11%. In summary, the proportion of cells satisfying the phenotype of the cell surface markers as CD34+CD43-KDR+CD73- reached 26.11% of the total cell number, that is, the overall differentiation efficiency was 26.11%. Close to or more than 90%, indicating that the differentiation is very uniform.

Example 6 The phenotype of the hematopoietic progenitor cells obtained in Example 5 was detected after CD34 enrichment

In this example, the total cell population obtained in Example 5 was sorted using magnetic beads targeting cell surface CD34 to enrich hematopoietic progenitor cells with CD34 expression on the cell surface, and CD34, CD43, KDR, CD73 antibodies were detected.

The results are shown in Figure 6, among the hematopoietic progenitor cells enriched by CD34, the proportion of CD34+ cells reached 98.57% after being enriched by CD34 (Figure 6A); the proportion of CD34+KDR+ cells was 97.58% (Figure 6B); CD34+ The proportion of CD43- cells was 94.72% (Figure 6C); the proportion of CD34+CD73- cells was 96.34% (Figure 6D). The above results further illustrate the homogeneity of the hematopoietic progenitor cell phenotype obtained in the present invention.

Example 7: Further refined detection of surface markers on the hematopoietic progenitor cells obtained in Example 6

In this example, the surface markers of the enriched hematopoietic progenitor cells obtained in Example 6 are further described. In particular, the cell phenotype was further refined using antibodies to the surface antigens DLL4 and CD184.

The results shown in Figure 7, among the hematopoietic progenitor cells enriched by CD34, the proportion of CD34+DLL4+ cells was 87.32% (Figure 7A), indicating that the phenotype of the hematopoietic progenitor cells obtained in the present invention can be further refined and defined as CD34+KDR+CD43-CD73-DLL4+. Further results showed that among the CD34-enriched hematopoietic progenitor cells, the proportion of CD34+CD184- cells was 71.92% (Figure 7B), indicating that the phenotype of the hematopoietic progenitor cells obtained in the present invention can be further refined and defined as CD34 +KDR+CD43-CD73-DLL4+CD184-.

Example 8: CHIR99021 treatment can significantly increase the proportion of CD34+KDR+ cells in EBs on day 5

When using T25 culture flasks for EB differentiation, other conditions and methods are as described above, and the concentration of CHIR99021 is gradient optimized, that is, cells are treated with 0uM, 6uM or 8uM CHIR99021 on D1-D3, and flow cytometry is used on D5 The expression of KDR and CD34 was determined by technique.

The result is shown in Figure 8. Compared with the control without CHIR99021 (CHIR99021 concentration is 0), CHIR99021 treatment significantly improved the differentiation efficiency of pluripotent stem cells to hematopoietic progenitor cells, the data expressed as the proportion of CD34+KDR+ cells in EBs was significantly improved after CHIR99021 treatment, The optimum was reached when the concentration of CHIR99021 was 6uM.

Example 9: SB431542 treatment can significantly increase the proportion of CD34+CD43- cells in EB on day 5

When using T25 culture flasks for EB differentiation, other conditions and methods are as described above, and the SB431542 treatment time is gradient optimized, that is, the cells are treated with 6uM SB431542 at D2-D4 for 48 hours, 36 hours, 24 hours or 0 hours, and Expression of CD34 and CD43 was determined on D5 using flow cytometry.

The result is shown in Figure 9. It can be seen that the processing time of SB431542 is crucial to the differentiation efficiency, and the relationship between the processing time and the differentiation efficiency presents a bell-shaped curve. Compared with the control without SB431542 (SB431542 treatment time was 0 hours), SB431542 treatment significantly increased the differentiation efficiency of pluripotent stem cells to hematopoietic progenitor cells, and the data were expressed as the proportion of CD34+CD43- cells in EBs after SB431542 treatment. Significant improvement was achieved, and the processing time of SB431542 reached the optimum at 24 hours.

Example 10: Optimizing the concentration of bFGF found that the concentration of bFGF affects the proportion of CD34+KDR+ cells in EB on the fifth day

When using T25 culture flasks for EB differentiation, other conditions and methods are as above, and the concentration of bFGF is gradient optimized, that is, 0ng/mL, 5ng/mL, 10ng/mL or 20ng/mL of bFGF are used for treatment on D1-D5 cells, and the expression of CD34 and KDR was measured at D5 using flow cytometry.

The results are shown in Figure 10. It can be seen that the effect of bFGF concentration on the differentiation efficiency presents a bell-shaped curve. When the bFGF concentration was 0, the differentiation efficiency was close to 0, and when the bFGF concentration was 20ng/mL, the differentiation efficiency decreased. The optimal concentration of bFGF is 10ng/mL.

Example 11: CHIR99021 and SB431542 treatment significantly increased the proportion of KDR+CD73- cells in the CD34+CD43- population

When using T25 culture flasks for EB differentiation, other conditions and methods are as above, D1-D3 use 6uM CHIR99021 to treat cells, D3-D4 use 6uM SB431542 to treat cells, and compare with the conditions without CHIR99021 and SB431542 treatment, in D5 The expression of CD34, CD43, KDR and CD73 was determined by flow cytometry.

The result is shown in Figure 11. Combined treatment with CHIR99021 and SB431542 significantly increased the proportion of CD34+CD43- cells (about 7 times) compared with no treatment with CHIR99021 and SB431542.

At the same time, more than 97% of the CD34+CD43- cells obtained without CHIR99021 and SB431542 treatment were KDR-, and only 57% of them were CD73-. 80% were KDR+, and more than 84% were CD73-, showing that a very homogeneous CD34+CD43-KDR+CD73- population was obtained.

Example 12: The ability of hematopoietic progenitor cells differentiated into CD43+CD45+ blood precursor cells after combined treatment with CHIR99021 and SB431542 is greatly improved

CD43 and CD45 are surface markers of mature blood progenitor cells. When using T25 culture flasks for EB differentiation, other conditions and methods are as above, D1-D3 use 6uM CHIR99021 to treat cells, D3-D4 use 6uM SB431542 to treat cells, and compare with the conditions without CHIR99021 and SB431542 treatment. In order to verify the ability of the obtained hematopoietic progenitor cells to further differentiate into blood precursor cells, the D5EB obtained by the above method was inoculated on OP9 trophoblast cells, and after continuing to culture for 7 days, the ratio of CD43 and CD45 in the suspension cells was detected.

The result is shown in Figure 12. The results showed that the ratio of hematopoietic progenitor cells treated with CHIR99021 and SB431542 to differentiate into CD43+CD45+ double-positive cells increased from 28.99% to 90.68%, an increase of more than 3 times, indicating that the hematopoietic progenitor cells obtained by combined treatment with CHIR99021 and SB431542 were differentiated into CD43+CD45+ double positive cells. The capacity of blood precursor cells is significantly improved.

Example 13: CD34+CD43-KDR+CD73- cells have the ability to differentiate into various blood cells

The blood cell clone formation assay can detect the differentiation ability of hematopoietic progenitor cells and prove that they can form different types of blood cell clones such as BFU-E, CFU-G/M/GM and CFU-GEMM.

When using T25 culture flasks for EB differentiation, other conditions and methods are as above, D1-D3 use 6uM CHIR99021 to treat cells, D3-D4 use 6uM SB431542 to treat cells, and the resulting EBs of D6, D7, D8, and D9 Digest into single cells for hemocyte colony formation experiments.

The results are shown in Figure 13. The results show that the hematopoietic progenitor cells of different days such as D6, D7, D8, and D9 obtained by the above method have the ability to form a variety of representative blood precursor cell clones, showing the stability of the method.

All documents mentioned in this application are incorporated by reference in this application as if each were individually incorporated by reference. In addition, it should be understood that after reading the above teaching content of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of the present application.

Claims (36)

1. A serum-free method for preparing hematopoietic progenitor cells, characterized in that, comprising steps: (a) providing pluripotent stem cells; (b) performing suspension culture on the pluripotent stem cells to form an embryoid body (embryoid body, EB); (c) in the presence of compound GSK-3β inhibitors, BMP-4, bFGF and VEGF, the embryoid body is induced to form mesoderm; (d) inducing culture of said mesoderm in the presence of compounds TGF-β inhibitor, BMP-4, bFGF and VEGF, thereby forming hematopoietic endothelial cells; and (e) transforming and culturing said hematopoietic endothelial cells in the presence of a combination of blood cell growth factors comprising SCF and TPO, thereby obtaining hematopoietic progenitor cells; Wherein the GSK-3β inhibitor is one or a combination of the following compounds: NP031112, TWS119, SB216763, CHIR-98014, AZD2858, AZD1080, SB415286, LY2090314 and CHIR99021; The TGF-β inhibitor is one of the following compounds or a combination thereof: A-83-01, GW6604, IN-1130, Ki26894, LY2157299, LY364947 (HTS-466284), LY550410, LY573636, LY580276, NPC-30345, SB-431542, SB-505124, SD-093, Sm16, SM305, SX-007, Antp-Sm2A, LY2109761. 2. The method according to claim 1, characterized in that, in step (a), comprising: digesting the pluripotent stem cells to form a single cell suspension. 3. The method according to claim 2, characterized in that, in step (a), the treatment is carried out with accutase digestive enzyme. 4. The method according to claim 1, characterized in that, in step (b), the seeding concentration of pluripotent stem cells is 0.1×10 ⁶ -5×10 ⁶ /mL. 5. the method for claim 1, is characterized in that, in step (b), in adding the CD34A of ROCK inhibitor, cultivate, the medium composition of described CD34A is as follows: basal medium, final concentration is 2~200μg/mL transferrin, final concentration 100~400μM MTG, final concentration 20~100μg/mL vitamin C, final concentration 0.5~5mM glutamine, final concentration 0.5~5μg/mL Recombinant human insulin. 6. The method according to claim 5, wherein the ROCK inhibitor is selected from the group consisting of Blebbistatin, HA-100, Y-27632, HA-1077, KD-025, Y-33075, Narciclasine or combination. 7. The method of claim 5, wherein in step (b), the ROCK inhibitor is Blebbistatin. 8. The method according to claim 5, characterized in that, in step (b), the time for suspension culture is 4-32 hours. 9. The method of claim 1, wherein the concentration of the GSK-3β inhibitor is 0.1-20 uM. 10. The method according to claim 1, characterized in that, in step (c), the culture time is 1-3.5 days. 11. The method according to claim 1, wherein in step (c), the BMP-4 concentration is 1-50ng/mL; the bFGF concentration is 0.5-50ng/mL; and the VEGF concentration is 1-50ng/mL; 100ng/mL. 12. The method of claim 1, wherein in step (d), the concentration of the TGF-β inhibitor is 0.1-10 uM. 13. The method according to claim 1, characterized in that, in step (d), the culture time is 0.5-4 days. 14. The method of claim 1, wherein in step (d), the concentration of BMP-4 is 0.5-50 ng/mL; the concentration of bFGF is 1-20 ng/mL; and the concentration of VEGF is 1-100 ng/mL mL. 15. The method according to claim 1, characterized in that, in step (e), bFGF and VEGF also exist in the culture system. 16. The method according to claim 1, characterized in that, in step (e), the culture system does not contain BMP-4. 17. The method according to claim 1, wherein the blood cell growth factor combination further comprises one or more blood growth factors selected from the group consisting of FLT3L, IL3, IL6, IGF1, IL11, IL7 , IL15. 18. The method of claim 1, wherein the combination of blood cell growth factors is selected from the group consisting of: 1) SCF, TPO; 2) SCF, TPO, FLT3L; 3) SCF, TPO, FLT3L, IL3, IL6; 4) SCF, TPO, FLT3L, IL3, IL6, IGF1; 5) SCF, TPO, FLT3L, IL3, IL6, IGF1, IL11; 6) SCF, TPO, FLT3L, IL3, IL6, IGF1, IL11, IL7; and 7) SCF, TPO, FLT3L, IL3, IL6, IGF1, IL11, IL7, IL15. 19. The method of claim 18, wherein the concentration of the blood cell growth factor is as follows: The concentration of SCF is 10-500ng/mL; TPO concentration is 10-300ng/mL; The concentration of FLT3L is 5-300ng/mL; The concentration of IL3 is 5-300ng/mL; The concentration of IL6 is 5-300ng/mL; The concentration of IGF1 is 10-100ng/mL; The concentration of IL11 is 5-200ng/mL; The concentration of IL7 is 1-200ng/mL; The concentration of IL15 was 5-200 ng/mL. 20. The method of claim 1, wherein in steps (c), (d) and (e), the culture is a suspension culture. 21. The method according to claim 1, further comprising: (f) detecting the cell phenotype of the formed hematopoietic progenitor cells. 22. The method according to claim 1, characterized in that the method is a method for preparing hematopoietic progenitor cells under serum-free conditions. 23. A population of hematopoietic progenitor cells, characterized in that, the population of hematopoietic progenitor cells is prepared by the method according to any one of claims 1-22. 24. The hematopoietic progenitor cell population according to claim 23, characterized in that, the hematopoietic progenitor cell population is formed by differentiation of pluripotent stem cells, wherein the proportion of CD34+ hematopoietic progenitor cells reaches more than 30%, wherein CD34+KDR Cells with a +CD43-CD73- phenotype accounted for more than 80% of CD34+ hematopoietic progenitor cells. 25. The population of hematopoietic progenitor cells according to claim 24, wherein the cells having a CD34+KDR+CD43-CD73- phenotype have a CD34+KDR+CD43-CD73-DLL4+ phenotype. 26. The population of hematopoietic progenitor cells according to claim 24, wherein the cells having a CD34+KDR+CD43-CD73- phenotype have a CD34+KDR+CD43-CD73-DLL4+CD184- phenotype. 27. The hematopoietic progenitor cell population according to claim 23, wherein the hematopoietic progenitor cell population is human hematopoietic progenitor cell population. 28. The hematopoietic progenitor cell population according to claim 24, wherein the pluripotent stem cells are human pluripotent stem cells, including human embryonic stem cells (ESC) and human induced pluripotent stem cells (iPSC). 29. The population of hematopoietic progenitor cells according to claim 23, wherein the population of hematopoietic progenitor cells has any one or more characteristics selected from the following: (i) more than 90% of the cells have the surface antigen CD34; (ii) More than 90% of the cells have the surface antigen combination CD34+KDR+; (iii) more than 90% of the cells have the surface antigen combination CD34+CD43-; (iv) More than 90% of the cells have the surface antigen combination CD34+CD73-; (v) more than 80% of the cells have the surface antigen combination CD34+DLL4+; and (vi) More than 70% of cells have surface antigen combination CD34+CD184-. 30. The population of hematopoietic progenitor cells according to claim 29, characterized in that, the population of hematopoietic progenitor cells has 3, 4, 5 or more than 6 characteristics. 31. The hematopoietic progenitor cell population according to claim 23, wherein the hematopoietic progenitor cell population has the ability to differentiate into CD43+CD45+ blood precursor cells; and/or The hematopoietic progenitor cell population has the ability to differentiate into erythroid blood cells; and/or The hematopoietic progenitor cell population has the ability to differentiate into myeloid blood cells; and/or The hematopoietic progenitor cell population also has the ability to differentiate into lymphocytes. 32. A pharmaceutical composition for treating blood diseases, said pharmaceutical composition comprising: an effective amount of the hematopoietic progenitor cell population according to claim 23, and a pharmaceutically acceptable carrier. 33. The pharmaceutical composition according to claim 32, wherein the pharmaceutical composition is a liquid preparation. 34. The pharmaceutical composition according to claim 33, wherein, in the pharmaceutical composition, the concentration of the hematopoietic progenitor cells is 1×10 ⁴ cells/mL-1×10 ⁶ cells/mL. 35. The pharmaceutical composition according to claim 34, wherein the concentration of the hematopoietic progenitor cells is 1×10 ⁵ cells/mL-9.9× ^{10 5} cells/mL. 36. A use of the hematopoietic progenitor cell population according to claim 23, characterized in that it is used for preparing a drug for treating blood diseases, and the blood diseases are selected from the group consisting of anemia, leukemia, or a combination thereof.

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