CN111607566A

CN111607566A - Method for differentiating human pluripotent stem cells into hematopoietic progenitor cells and application thereof

Info

Publication number: CN111607566A
Application number: CN201910133886.XA
Authority: CN
Inventors: 陈涛涛; 俞君英; 张颖; 周桃
Original assignee: Nuwacell Ltd
Current assignee: Nuwacell Ltd
Priority date: 2019-02-22
Filing date: 2019-02-22
Publication date: 2020-09-01
Anticipated expiration: 2039-02-22
Also published as: CN111607566B

Abstract

The invention provides a method for differentiating human pluripotent stem cells into hematopoietic progenitor cells and application thereof. The invention also provides hematopoietic progenitor cells formed from pluripotent stem cells differentiated and having the phenotype CD34+ KDR + CD43-CD 73-. The method of the invention can efficiently and rapidly prepare the hematopoietic progenitor cells with stable differentiation effect under the serum-free condition, and the hematopoietic progenitor cells have the differentiation capacity of erythroid, myeloid and lymphoid systems.

Description

Method for differentiating human pluripotent stem cells into hematopoietic progenitor cells and application thereof

Technical Field

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

Background

Hematological disorders are diseases that originate in the hematopoietic system or affect the hematopoietic system with abnormal changes in the blood, which often manifest as anemia, bleeding, fever, and the like.

The incidence of malignant cancers of children in China is on the rise, and data in 2014 shows that the incidence of leukemia in the malignant tumors of children is the first and accounts for about one third. The clinical chemotherapy effect is often not ideal for malignant hematological diseases. Since the first development of HSC (hematopoietic stem cell) transplantation by professor Thomas in the middle of the twentieth century, HSC transplantation has been widely used in clinical treatment of leukemia, and has become one of the effective means for treating acute leukemia, malignant lymphoma, severe aplastic anemia, and the like.

Currently HSCs are mainly derived from cord blood, bone marrow and peripheral blood. HSC transplantation is mainly divided into autologous and allogeneic HSC transplantation. Although autografting has the advantages of no graft rejection, no graft-versus-host disease, and other complications, the number of autologous HSCs stored in the cord blood bank is short-lived, limiting their clinical use in disease. Although long-term efficacy is superior to that of autologous transplantation and recurrence rate is low, allogenic transplantation has extremely low efficiency and limited sources, thereby limiting clinical application of allogeneic HSC transplantation.

Therefore, there is an urgent need in the art to find a safer, less costly, and stable source of HSC resources. 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 (ESCs) and human Induced Pluripotent Stem Cells (iPSCs).

Research shows that ESCs of mouse, monkey and human can be induced to differentiate into various blood cells in vitro, but ESCs are derived from embryos at early development stage and have the problems of difficult material taking, immunological rejection, ethical morality and the like.

Human induced pluripotent stem cells (ipscs) can be reprogrammed in vitro from somatic cells such as human skin, blood, etc., and have an unlimited proliferation capacity similar to ESCs, and the ability to differentiate in vitro into almost all functional cells, including hematopoietic stem cells. The iPSC characteristic successfully avoids two most key problems of immunological rejection and ethicity, and provides possibility for clinical transplantation of clinically obtained in vitro hematopoietic stem cells.

There have been many studies reporting methods for 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 using an embryoid body differentiation method with exogenous addition of a mixture of BMP-4 and cytokines (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 (otherwise known as trophoblast cells) (Ledran MH et al, 2008).

Yu C et al reported a method of inducing 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 have only the ability to differentiate into erythroid and myeloid blood cells, but not into lymphoid blood cells (i.e., T cells, B cells, and NK cells), and thus have no ability to reconstitute the entire hematopoietic system.

Kennedy M et al obtained mature hematopoietic progenitor cells with T cell differentiation ability under serum-free and stromal cell-free conditions, and demonstrated that the mature hematopoietic progenitor cells were obtained via the Hematogenous Endothelium (HE) stage with a surface marker combination of CD34+ CD43-, and their function was evaluated by T cell differentiation ability (Kennedy M et al, 2013).

Activation of the Notch signaling pathway was found to be important in the process of forming mature HE by Uenishi GI et al, 2015.

Although the art is aware of the process of in vitro differentiation of human pluripotent stem cells into hematopoietic progenitor cells, the existing methods of differentiation have some disadvantages, including unstable differentiation efficiency, or the use of serum-containing culture systems or trophoblast cells, which is not suitable for the production of subsequent clinical-grade cell preparations.

Therefore, there is an urgent need in the art to develop a method for efficiently and rapidly preparing hematopoietic progenitor cells that are stably differentiated under serum-free conditions.

Disclosure of Invention

The invention aims to provide a method for preparing hematopoietic progenitor cells with stable differentiation efficiently, quickly and in a serum-free manner

In a first aspect of the invention, there is provided a hematopoietic progenitor cell which is formed by differentiation of pluripotent stem cells and has the phenotype CD34+ KDR + CD43-CD 73-.

In another preferred embodiment, the pluripotent stem cells are selected from the group consisting of: embryonic Stem Cells (ESCs), Induced Pluripotent Stem Cells (iPSCs), or a combination thereof.

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

In another preferred embodiment, the hematopoietic progenitor cells have the phenotype CD34+ KDR + CD43-CD73-DLL4+ CD 184-.

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

In another preferred embodiment, the pluripotent stem cell is a human pluripotent stem cell, and comprises a human Embryonic Stem Cell (ESC) and a human Induced Pluripotent Stem Cell (iPSC).

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

In another preferred embodiment, the hematopoietic progenitor cells have any one or more characteristics selected from the group (a) below:

(i) more than 90% of the cells have the surface antigen CD 34;

(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+ CD 43-;

(iv) more than 90% of the cells have the surface antigen combination CD34+ CD 73-;

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

(vi) more than 70% of the cells have the surface antigen combination CD34+ CD 184-.

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

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

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

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

In another preferred embodiment, said hematopoietic progenitor cells further have the ability to differentiate into lymphocytes.

In a second aspect of the present invention, there is provided a pharmaceutical composition for treating hematological disorders, said pharmaceutical composition comprising: an effective amount of a hematopoietic progenitor cell according to the first aspect of the invention, and a pharmaceutically acceptable carrier.

In another preferred embodiment, the pharmaceutical composition is a liquid formulation.

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

In another preferred embodiment, the pharmaceutical composition is an intravenous agent.

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

In another preferred embodiment, the hematopoietic progenitor cell concentration is 1 × 10³Per ml-1 × 10⁷One/ml, preferably 1 × 10⁴-1×10⁶One/ml, more preferably 1 × 10⁵Per ml-9.9 × 10⁵One per ml.

In another preferred embodiment, the hematological disorder is selected from the group consisting of: anemia, leukemia, or a combination thereof.

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

(a) providing a pluripotent stem cell;

(b) performing suspension culture on the pluripotent stem cells to form Embryoid Bodies (EBs);

(c) inducing culture of said embryoid bodies in the presence of a compound GSK-3 β inhibitor, thereby forming mesoderm;

(d) (ii) inducing culture of said mesoderm in the presence of a compound TGF- β inhibitor, thereby forming hematogenic endothelial cells;

(e) transforming said hematopoietic endothelial cells in the presence of a combination of blood cell growth factors to obtain hematopoietic progenitor cells according to the first aspect of the invention.

In another preferred example, in the step (a), the method comprises: subjecting the pluripotent stem cells to a digestion process, thereby forming a single cell suspension.

In another preferred embodiment, the treatment is performed with accutase digestive enzyme.

In another preferred embodiment, in step (b), the pluripotent stem cells are seeded at a concentration of 0.1 × 10⁶-5× 10⁶/ml。

In another preferred embodiment, in step (b), the culture is performed in a maintenance medium for CD34A or pluripotent stem cells supplemented with a ROCK inhibitor or other compound that promotes the survival of individual human pluripotent cells.

In another preferred embodiment, the ROCK inhibitor or other compound that promotes the survival of a single human pluripotent cell is selected from the group consisting of: blebbistatin, HA-100, Y-27632, HA-1077, KD-025, Y-33075, Narcilase or a combination thereof.

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

In another preferred example, in step (b), the suspension culture is carried out for 4 to 24 hours, 8 to 24 hours, 12 to 24 hours, 16 to 24 hours, 4 to 32 hours, 8 to 32 hours, 12 to 32 hours, 16 to 32 hours, or 12 to 24 hours.

In another preferred embodiment, in step (c), the GSK-3 β inhibitor may be one of the following compounds or a combination thereof: NP031112, TWS119, SB216763, CHIR-98014, AZD2858, AZD1080, SB415286, LY2090314, and CHIR 99021.

In another preferred embodiment, in step (c), the GSK-3 β inhibitor may be CHIR 99021.

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

In another preferred example, in step (c), the culturing period is 1 to 3.5 days, 1.5 to 3.5 days, 2 to 3.5 days, 1 to 3 days, 1.5 to 3 days, or 2 to 3 days.

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

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

In another preferred embodiment, in step (d), the TGF- β inhibitor may be one of the following compounds or a combination thereof: a-83-01, GW6604, IN-1130, Ki26894, LY2157299, LY364947 (HTS-466284), LY550410, LY 573636, LY580276, NPC-30345, SB-431542, SB-505124, SD-093, Sm16, SM305, SX-007, Antp-Sm2A, LY 2109761.

In another preferred example, in step (d), the TGF- β inhibitor may be SB 431542.

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

In another preferred example, in step (d), the culturing time may be 0.5 to 1 day, 0.5 to 2 days, 0.5 to 3 days, 0.5 to 3.5 days, 0.5 to 4 days, or 1 to 2 days.

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

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

In another preferred embodiment, in step (e), bFGF and VEGF are also present in the culture system.

In another preferred embodiment, in step (e), BMP-4 is not contained in said culture system.

In another preferred embodiment, in step (e), the combination of blood cell growth factors comprises SCF and TPO.

In another preferred embodiment, 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, or a combination thereof.

In another preferred embodiment, the blood cell growth factor combination 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, IL 7; and

7)SCF、TPO、FLT3L、IL3、IL6、IGF1、IL11、IL7、IL15。

in another preferred embodiment, the concentration of each blood cell growth factor is as follows:

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

the concentration of TPO is 10-300ng/mL, and the preferable concentration can be 100 ng/mL;

the concentration of FLT3L is 5-300ng/mL, and the preferable concentration can be 50 ng/mL;

IL3 concentration is 5-300ng/mL, preferably concentration can be 50 ng/mL;

IL6 concentration is 5-300ng/mL, preferably concentration can be 50 ng/mL;

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

IL11 concentration is 5-200ng/mL, preferably concentration can be 10 ng/mL;

IL7 concentration is 1-200ng/mL, preferably concentration can be 10 ng/mL;

IL15 may be present at a concentration of 5-200ng/mL, preferably at a concentration of 20 ng/mL.

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

In another preferred example, the method further comprises:

(f) and (4) detecting the cell phenotype of the formed hematopoietic progenitor cells.

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

In a fourth aspect of the invention, there is provided the use of a hematopoietic progenitor cell of the first aspect of the invention in the manufacture of a medicament for the treatment of a hematological disorder.

In another preferred embodiment, the medicament is a liquid preparation.

In a fifth aspect of the invention, there is provided a method of treating a hematological disorder, comprising the steps of: administering to a subject in need thereof a hematopoietic progenitor cell according to the first aspect of the invention, or a pharmaceutical composition comprising a hematopoietic progenitor cell.

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

In another preferred embodiment, the site of administration is within a vein or bone marrow of said subject.

It is to be understood that within the scope of the present invention, the above-described features of the present invention and those specifically described below (e.g., in the examples) may be combined with each other to form new or preferred embodiments. Not to be reiterated herein, but to the extent of space.

Drawings

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

FIG. 2 shows an embryoid body formed from an iPSC in one embodiment of the present invention.

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

FIG. 4 shows the induction of differentiation of mesodermal cells to the Hematogenic Endothelium (HE) in one embodiment of the invention.

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

FIG. 6(6A-6D) shows the results of the characterization of the cell phenotype after enrichment of hematopoietic progenitor cells in one embodiment of the invention.

FIG. 7(7A-7B) shows the results of further refinement of the hematopoietic progenitor phenotype identified in one embodiment of the present invention.

FIG. 8(8A-8C) shows a comparative experiment with known methods during optimization of the present technology. Compared with the known method without CHIR99021 (concentration of CHIR99021 is 0), the treatment of CHIR99021 significantly improves the efficiency of differentiation of pluripotent stem cells into hematopoietic progenitor cells, and the data shows that the proportion of CD34+ KDR + cells in EB is significantly increased after the treatment of CHIR99021, and is optimal when the concentration of CHIR99021 is 6 uM.

FIG. 9 shows another comparative experiment with the known method in the optimization process of the present invention. SB431542 treatment significantly improved the efficiency of differentiation of pluripotent stem cells into hematopoietic progenitors compared to the method without SB431542 (0 hour treatment time for SB 431542), data indicating that the proportion of CD34+ CD 43-cells in EB was significantly increased after SB431542 treatment, and was optimized at 24 hours treatment time for SB 431542.

FIG. 10 shows the optimization of bFGF concentration in the process during the optimization of the present invention. The data show the effect of bFGF on the proportion of CD34+ KDR + cells in EB.

FIG. 11 shows another comparative experiment with another method in the optimization of the present technique. The data show that combined treatment with CHIR99021 and SB431542 significantly increased the proportion of KDR + CD 73-cells in the CD34+ CD 43-population compared to treatment without CHIR99021 and SB431542, resulting in a more uniform phenotype of the differentiated hematopoietic progenitor cells. In the figure, CHIR denotes CHIR99021, and SB denotes SB 431542.

FIG. 12 shows that CD34+ CD43-KDR + CD 73-cells can differentiate efficiently into CD43+ CD45+ blood precursor cells. In the figure, CHIR denotes CHIR99021, and SB denotes SB 431542.

FIG. 13 shows that KDR + CD34+ CD43-CD 73-cells have the ability to differentiate into various blood cells.

Detailed Description

The present inventors have made extensive and intensive studies and have found, for the first time, that it is possible to efficiently and rapidly produce hematopoietic progenitor cells having a stable differentiation effect in the presence of a specific compound under serum-free conditions by optimizing the conditions, and that the hematopoietic progenitor cells have a differentiation ability of both erythroid, myeloid and lymphoid lineages. The present invention has been completed based on this finding.

Term(s) for

As used herein, the terms "above" and "below" include present numbers, e.g., "95% or more" means 95% or more and "0.2% or less" means 0.2% or less.

The term "pluripotency" 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 gastric wall, gastrointestinal tract, lung), mesoderm (muscle, bone, blood, genitourinary) or ectoderm (epidermal tissue and nervous system).

"pluripotent stem cells" refers to cells that are capable of producing all three germinal cells, i.e., endoderm, mesoderm, and ectoderm. While it is theorized that pluripotent stem cells can differentiate into any cell of the body, pluripotency experimental assays are generally based on the differentiation of pluripotent cells into several cell types per germinal layer.

The term "induced pluripotent stem cell", often abbreviated as iPS cell or iPSC, refers to a pluripotent stem cell such as a muscle cell, neuron, epidermal cell or the like, which is artificially prepared from a non-pluripotent cell (usually an adult somatic cell) or a terminally differentiated cell (e.g., fibroblast, hematopoietic cell) by introducing or contacting a reprogramming factor.

The term "embryonic stem cell", often abbreviated ES, is a pluripotent stem cell derived from an early embryo.

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

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

The term "embryoid body," i.e., an embryoid body or aggregate, refers to a homogeneous or heterogeneous cell cluster comprising differentiated cells, partially differentiated cells, and/or pluripotent stem cells cultured in suspension. To summarize some of the cues inherent to in vivo differentiation, certain aspects of the invention may use a three-dimensional embryoid body as an intermediate step. At the onset of cell aggregation, differentiation can be initiated and cells can begin to reproduce embryonic development to a limited extent. Although they are unable to form trophectoderm tissue, almost all other types of cells present in an organism can develop. The present invention can further promote the differentiation of hematopoietic progenitor cells after the formation of embryoid bodies.

As used herein, the term "hematologic disorder" refers to a disease associated with cytopathic effects in the blood, representative hematologic disorders include (but are not limited to): anemia, thrombocytopenia, leukemia, lymphoma, severe aplastic anemia, multiple myeloma.

As used herein, the term "CHIR 99021" refers to a compound having a CAS No. 252917-06-9, with the understanding that this term also includes CHIR99021 and salts, especially pharmaceutically acceptable salts, thereof. The chemical structure of CHIR99021 is:

as used herein, the term "SB 431542" refers to compounds having a CAS No. 301836-41-9. It is to be understood that the term also includes SB431542 and salts thereof, especially pharmaceutically acceptable salts. The specific chemical structure is as follows:

preparation method

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

The method of the invention is mainly characterized by comprising the following steps: (1) performing mesoderm induction by using a small molecule compound CHIR99021 so as to obtain rapid and uniform mesoderm; (2) induction of Hematogenic Endothelium (HE) using SB431542, thereby removing the effects of primitive hematopoiesis; (3) the cell phenotype of the final product prepared by the method is CD34+ KDR + CD43-CD73-, and the final product is mature hematopoietic progenitor cells with high-efficiency T cell differentiation capacity.

In the invention, in addition to the specific small molecule compounds CHIR99021 and SB431542, the conditions such as the concentration, the culture time and the like of the small molecule compounds are optimized at different culture stages, so that the optimized method not only can quickly and efficiently prepare the hematopoietic progenitor cells, but also can stably differentiate the hematopoietic progenitor cells into a plurality of different blood cells (including cells with erythroid, myeloid and lymphoid lineages).

It will be appreciated that in the method of the invention, in addition to using the specific small molecule compounds CHIR99021 and SB431542 at different stages of culture, media and culture components known in the art may be used. To meet the requirements of clinical-grade cell preparations, the culture medium or culture system of the invention is preferably serum-free. In addition, other components beneficial for inducing differentiation, such as other small molecule compounds, including additional GSK3beta inhibitors and TGFbeta inhibitors, may be additionally added to the medium or culture system.

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

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

Hematopoietic progenitor cells

The term "hematopoietic progenitor cells of the invention", as used herein, refers to hematopoietic progenitor cells formed by directed induced differentiation from pluripotent stem cells, as described in the first aspect of the invention. Unless otherwise indicated, "hematopoietic progenitor cell" refers to a cell having the phenotypic characteristics of CD34+ KDR + CD43-CD 73-. A particularly preferred hematopoietic progenitor cell is a CD34+ KDR + CD43-CD 73-purified population of cells.

The hematopoietic progenitor cells of the present invention have the ability to differentiate into erythroid, myeloid and lymphoid lineages.

The invention also provides cell preparations comprising the hematopoietic progenitor cells of the invention. The cell preparation can be used for treating hematopathy such as leukemia.

One of ordinary skill in the art can use, treat, administer, etc., the hematopoietic progenitor cells using conventional methods. Such as: before each batch of hematopoietic progenitor cells is released or used, it must be checked for sterility, endotoxin and mycoplasma, and DNA identity. The cells distributed in each batch are required to meet the conditions that the cell activity is more than or equal to 95 percent and the cell purity (the positive index is more than or equal to 95 percent and the negative index is less than 2 percent). The detection results of acute toxicity and allergy of the hematopoietic progenitor cells are negative.

Antigen detection of hematopoietic progenitor cells

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

The CD34 antigen is a highly glycosylated single-pass transmembrane protein that is selectively expressed on the surface of human Hematopoietic Stem Cells (HSCs), Hematopoietic Progenitor Cells (HPCs) and vascular Endothelial Cells (ECs). In the present invention, the proportion of CD 34-bearing hematopoietic progenitor cells in the total cell population is preferably greater than or equal to 90% after purification and enrichment.

KDR antigen is a Vascular Endothelial Growth Factor (VEGF) receptor, widely expressed in various mesodermal tissues during development, and expressed in vascular endothelial cells at the embryonic stage. In vitro and in vivo data show that KDR is critical for the development of vascular endothelial and hematopoietic cells. In the present invention, the proportion of the hematopoietic progenitor cells with KDR in the total cell population after purification and enrichment is preferably more than or equal to 90%.

The CD43 antigen is a glycoprotein encoded by the SNP gene, also known as leukocyte sialoglycoprotein or sialoprotein, expressed on the surface of most blood leukocytes such as B cells, T cells, NK cells, granulocytes. In the present invention, hematopoietic progenitor cells differentiated from ipscs were labeled using the feature of being negative to CD 43. In the present invention, the proportion of CD 43-negative hematopoietic progenitor cells in the total cell population is preferably greater than or equal to 90% after purification and enrichment.

The CD73 antigen is an extracellular-5' nucleotidase and is also a signal transduction molecule expressed on T cells and subpopulations of B cells, epithelial cells, endothelial cells and mesenchymal stem cells. In the present invention, hematopoietic progenitor cells differentiated from pluripotent stem cells (e.g., ipscs) are labeled using the feature of being negative for CD 73. In the present invention, the proportion of CD 73-negative hematopoietic progenitor cells in the total cell population is preferably greater than or equal to 90% after purification and enrichment.

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

CD184, also known as CXCR4, is a G protein-coupled chemokine receptor with a 7-transmembrane structure. CD184 is expressed predominantly on the surface of resting T cells. In the present invention, CD 184-negativity (non-expression) is used as one of the characteristics characterizing the phenotype of hematopoietic progenitor cells. In the present invention, the proportion of CD 184-negative hematopoietic progenitor cells in the total cell population after purification and enrichment is preferably greater than or equal to 70%.

The purity and degree of differentiation of the hematopoietic progenitor cells of the invention can be measured using conventional methods, such as flow cytometry. When detecting, different specific antibodies which are specific to corresponding cell surface antigens are added, and the antibodies can be complete monoclonal or polyclonal antibodies or antibody fragments with immunological activity, such as Fab' or (Fab)₂A fragment; single chain Fv molecules (scFV); or a chimeric antibody. The antibody is added to bind to the antigen on the cell surface for a period of time, and the cells can be automatically analyzed and/or sorted using a flow cytometer.

Pharmaceutical composition and application thereof

The invention also provides a pharmaceutical composition comprising an effective amount of the hematopoietic progenitor cells induced to differentiate from pluripotent stem cells of the invention, and a pharmaceutically acceptable carrier.

Typically, the hematopoietic progenitor cells of the invention are formulated in a non-toxic, inert, and pharmaceutically acceptable aqueous carrier medium, such as physiological saline, at a pH of typically about 6 to about 8, preferably about 6.5 to about 7.5.

As used herein, the term "effective amount" or "effective dose" refers to an amount that produces a function or activity in, and is acceptable to, a human and/or an animal.

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

The pharmaceutical composition of the present invention contains carriers including (but not limited to): saline, buffer, glucose, water, glycerol, ethanol, and combinations thereof. The pharmaceutical preparation is usually adapted to the administration mode, and the pharmaceutical composition of the present invention can be prepared in the form of injection, for example, by a conventional method using physiological saline or an aqueous solution containing glucose and other adjuvants. The pharmaceutical composition is preferably manufactured under sterile conditions. The amount of active ingredient administered is a therapeutically effective amount. The pharmaceutical preparation of the invention can also be prepared into a sustained release preparation.

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

The pharmaceutical composition of the present invention is preferably an intravenous agent. In another preferred embodimentWherein the concentration of said hematopoietic progenitor cells in said intravenous agent is 1 × 10³Per ml-1 × 10⁷One/ml, preferably 1 × 10⁴-1×10⁶One/ml, more preferably 1 × 10⁵Per ml-9.9 × 10⁵One per ml.

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

In the present invention, hematopoietic progenitor cells are administered to a subject in need thereof, preferably intravenously, to treat the corresponding hematological disorder.

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

The main advantages of the invention include:

firstly, the hematopoietic progenitor cells obtained by the method have the capacity of differentiating into lymphoid blood cells including T cells, and have great clinical application potential;

secondly, the differentiation process is finely regulated by using a small molecular compound, so that stable and efficient differentiation is realized, and the proportion of CD34 positive hematopoietic progenitor cells in a final culture system can reach more than 30%; wherein the phenotype of CD34+ KDR + CD43-CD 73-cells accounts for more than 80% of CD34+ cells, and the phenotype of CD34+ KDR + CD43-CD 73-cells has strong capacity of differentiating into T cells;

thirdly, the whole differentiation process uses a serum-free culture medium and does not use trophoblast cells, so that the method is suitable for the production of subsequent clinical-grade cell preparations;

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

The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Experimental procedures without specific conditions noted in the following examples, generally followed by 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 recommendations. Unless otherwise indicated, percentages and parts are by weight.

Material

The composition of the CD34A medium was as follows:

serial number	Composition (I)	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	Basic culture medium

Preferably. The culture medium is a serum-free culture medium and has definite chemical components. In addition, transferrin, recombinant human insulin, 1-thioglycerol, vitamin C, glutamine, and other components can be added into the basic culture medium.

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

MTG refers to the organic compound 1-Thioglycerol (1-Thioglycerol), CAS No.96-27-5, with molecular formula HSCH2CH (OH) CH2 OH.

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

Glutamine refers to L-Glutamine (L-Glutamine), which is a coding amino acid in protein synthesis, a non-essential amino acid for mammals, and is an essential additive for cell culture in the present invention.

The recombinant human insulin refers to recombinant human insulin protein produced by recombinant DNA technology, and the biological activity is the same as that of natural human insulin protein.

Basal media, among chemically defined media, include Basal cell culture media such as Iscove's modified Dulbecco's Medium (IMDM broth), Eagle's Basic Medium (BME), Eagle MEM, DMEM, Ham, RPMI1640, and Fischer Medium, variants or combinations thereof.

Example 1

Formation of Embryoid Bodies (EB)

In this example, ipscs cultured to have a degree of polymerization of 90% and in a well-differentiated state were digested into a single cell suspension, which was then resuspended in human pluripotent stem cell medium at a certain density, and the suspension was placed on a shaker in a 37 ℃ incubator and cultured overnight with shaking, to form Embryoid Bodies (EBs) with uniform size and morphology. The experimental method is as follows:

1.1. culture of human iPSC

The human ipscs used in this example were subjected to strict pluripotency validation (expressing various pluripotency markers and forming teratomas comprising three germ layers, inner, middle and outer, in immunodeficient mice). The ipscs are normally cultured in iPSC maintenance medium, and the medium used is E8 or TeSR or other similar medium.

1.2 formation of EB

An embryoid body formation experiment was performed when ipscs were cultured to 90% degree of polymerization as described above. The specific operation is as follows: the pluripotent stem cells are dissociated into substantially individual cells using mechanical or enzymatic methods known in the art, for example, accutase digestive enzymes digest ipscs into a complete single cell suspension and the dispersed pluripotent cells are seeded at a density of about 0.1 million to about 5million/mL in CD34A or pluripotent stem cell maintenance medium. In certain aspects, a ROCK inhibitor or other small molecule compound effective to increase cloning efficiency and cell survival, such as Blebbistatin, HA-100, Y-27632, HA-1077, KD-025, Y-33075, Narcilase, or a combination thereof, can be added to the culture medium at an effective concentration. 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 12.5uM, or any concentration range therein. The cell suspension is placed in a 37 ℃ incubator and incubated in a non-static manner, such as shaking, spinning or stirring, such as a high capacity bioreactor, to maintain the cells in any culture at a controlled rate of movement. Agitation can improve circulation of nutrients and cellular waste, and can also provide a more uniform environment to control cell aggregation. For example, the rotational speed may be set to about 6, 15, 30, 40, 50, 60, 70, 80, 90, 100rpm, or any range therein; the incubation period for this step of non-static culture may 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 of more uniform size and morphology are obtained, and the diameter of the cell embryoid bodies can be about 50, 65, 75, 85, 95, 105, 115, 125, 135, 145, 155, 170, 185, 200uM, or any range of diameters therein. The diameter may be an average diameter, a median or an average diameter. In another aspect, EB can comprise at least or about 10, 55, 95, 135, 175, 225, 550, 750, 1000 cells, or any range derivable therein, in at least about 25%, 35%, 45%, 55%, 70%, 85%, 95%, 99%, or any range of ratios therein.

In this example, T25 flask was used, and the concentration of Blebbistatin was 2.5 uM; the incubation time was 10 hours. At the end of the culture, the EB had a diameter of about 100uM (FIG. 2).

Example 2

Differentiation of embryoid bodies into mesoderm

In this example, the EB from D1 was resuspended in fresh CD34A medium supplemented with BMP-4, bFGF and VEGF, and the GSK3 β inhibitor CHIR99021 was added to the medium at an optimal concentration, and the medium was further cultured with shaking on a shaker in a 37 ℃ incubator for 2 days, so that the EB volume continued to increase, and more uniform mesodermal differentiation was obtained. The experimental method is as follows:

the flask containing D1EB (example 1) was removed from the incubator, the flask was tilted to allow the EB to settle to the bottom, the supernatant was removed, washed once with IMDM basal medium, and then fresh CD34A medium was added, along with the cytokines BMP-4, bFGF, VEGF, and the small molecule compound CHIR 99021.

In this example, a T25 flask was used for the culture, in which the concentration of BMP-4 was 1 ng/mL; the concentration of bFGF is 0.5 ng/mL; the concentration of VEGF was 5 ng/mL. Concentration of CHIR99021 was 4uM at the end of the culture, the EB was translucent and nearly spherical with a diameter between 150uM and 200uM (FIG. 3).

Example 3

Induction of mesodermal cell differentiation into Hematogenic Endothelium (HE)

In this example, the EBs prepared in example 2 on the third day (D3) were resuspended in fresh CD34A medium supplemented with BMP-4, bFGF and VEGF and the optimized concentration of the TGF β inhibitor SB431542 was added to the medium and continued to be cultured on a shaker in a 37 ℃ incubator for 1 day with continued shaking, the EB volume continued to increase, inducing the differentiation of mesodermal cells into HE cells.

The experimental method is as follows: the flask containing the EB of D3 was removed from the incubator, the flask was tilted to sink the EB to the bottom, the supernatant was removed, washed once with IMDM basal medium, and then fresh CD34A medium was added, along with the cytokines BMP-4, bFGF, VEGF, and the small molecule compound SB 431542.

In this example, a T25 flask was used for the culture, in which the concentration of BMP-4 was 0.5 ng/mL; the concentration of bFGF is 1 ng/mL; the concentration of VEGF was 5 ng/mL. The concentration of SB431542 was 6 uM. The treatment time was 24 hours. At the end of the culture, EBs were translucent and approximately spherical with diameters between 150-300 um (FIG. 4).

Example 4

Induction of Endothelial-Hematopoietic transformation (EHT)

In this example, day 4 (D4) EBs prepared in example 3 were resuspended in fresh CD34A media supplemented with VEGF and bFGF and blood cell growth factor combinations (including but not limited to SCF, TPO, FLT3L, IL3, IL6, IGF1, IL11) at optimized concentrations were added to the media and continued to shake culture on a shaker in a 37 ℃ incubator for 1-6 days to induce EHT process to generate CD34 positive hematopoietic progenitor cells.

The experimental method is as follows:

the flask containing D4EB was removed from the incubator, the flask was tilted to allow the EB to settle to the bottom, the supernatant removed, washed once with IMDM basal medium, and then fresh CD34A medium was added, along with the cytokines bFGF, VEGF, and blood cell growth factor combination. In this example, the bFGF concentration is 2 ng/mL; VEGF concentration was 5 ng/mL. There may be various combinations of blood cell growth factors, for example:

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, cell phenotype was detected by flow cytometry using antibodies against CD34, CD43, CD73 and KDR.

In this example, all cells from day five of differentiation were examined, including non-blood cells, non-hematopoietic vascular endothelial precursor cells, hematopoietic progenitor cells, and the like.

The results show that the phenotype of the resulting cells is: CD34+ CD43-KDR + CD 73-; wherein the proportion of CD34+ cells to total cells reached 33.21% (fig. 5A); the proportion of CD 43-cells accounted for CD34+ cells reached 91.75% (fig. 5B), thus the proportion of CD34+ CD 43-cells accounted for the total number of cells was 33.21% X91.75% — 30.47%; the proportion of KDR + cells to CD34+ CD 43-cells was 97.13% (fig. 5C), thus the proportion of CD34+ CD43-KDR + cells to total cells was 30.47% X97.13% ═ 29.60%; the proportion of CD 73-cells to CD34+ CD43-KDR + cells was 88.21% (fig. 5D), whereas the proportion of CD34+ CD43-KDR + CD 73-cells to the total number of cells was 29.60% X88.21% ═ 26.11%. In conclusion, the proportion of the cells satisfying the cell surface marker phenotype of CD34+ CD43-KDR + CD 73-reaches 26.11% of the total cell number proportion, namely the total differentiation efficiency is 26.11%, and meanwhile, the coincidence rate of the four surface markers is close to or exceeds 90%, which indicates that the differentiation is very uniform.

Example 6 enrichment of hematopoietic progenitor cells obtained in example 5 with CD34 and detection of their phenotype

In this example, the total cell population obtained in example 5 was sorted using magnetic beads against cell surface CD34 to enrich for hematopoietic progenitor cells with CD34 expression on the cell surface and detected using antibodies to CD34, CD43, KDR, CD 73.

The results are shown in fig. 6, in which the hematopoietic progenitor cells enriched with CD34, after enrichment with CD34, the proportion of CD34+ cells reached 98.57% (fig. 6A); the proportion of CD34+ KDR + cells was 97.58% (fig. 6B); the proportion of CD34+ CD 43-cells was 94.72% (fig. 6C); the proportion of CD34+ CD 73-cells was 96.34% (FIG. 6D). The above results further illustrate the uniformity of the phenotype of hematopoietic progenitor cells obtained in accordance with the present invention.

Example 7: further detailed examination of surface markers for hematopoietic progenitor cells obtained in example 6

In this example, the surface markers for enriched hematopoietic progenitor cells obtained in example 6 are further described. In particular, antibodies to the surface antigens DLL4 and CD184 were used to further refine the cell phenotype.

The results are shown in fig. 7, in which the cell ratio of CD34+ DLL4+ in the CD 34-enriched hematopoietic progenitor cells is 87.32% (fig. 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 the cell ratio of CD34+ CD 184-in CD 34-enriched hematopoietic progenitor cells was 71.92% (FIG. 7B), suggesting 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+ CD 184-.

Example 8: the CHIR99021 treatment can obviously improve the proportion of CD34+ KDR + cells in EB at the 5 th day

When EB differentiation was performed using T25 flasks, other conditions were as described above, gradient optimization of concentration of CHIR99021, i.e., treating cells with 0uM, 6uM or 8uM of CHIR99021 at D1-D3, respectively, and measuring the expression of KDR and CD34 at D5 using flow cytometry.

The results are shown in FIG. 8. Compared with the control without CHIR99021 (concentration of CHIR99021 is 0), the treatment of CHIR99021 significantly improves the efficiency of differentiation of pluripotent stem cells into hematopoietic progenitor cells, and the data shows that the proportion of CD34+ KDR + cells in EB is significantly increased after the treatment of CHIR99021, and is optimal when the concentration of CHIR99021 is 6 uM.

Example 9: SB431542 treatment significantly increased the proportion of CD34+ CD 43-cells in day 5 EBs

When EB differentiation was performed using T25 flasks, other conditioning methods were as described above, gradient optimization of SB431542 treatment time, i.e., cells were treated with 6uM SB431542 at D2-D4 for 48 hours, 36 hours, 24 hours or 0 hours, respectively, and CD34 and CD43 expression was determined at D5 using flow cytometry.

The results are shown in FIG. 9. It can be seen that the treatment time of SB431542 is critical to the differentiation efficiency, and the relationship between the treatment time and the differentiation efficiency shows a bell-shaped curve. SB431542 treatment significantly improved the efficiency of differentiation of pluripotent stem cells into hematopoietic progenitors compared to controls without SB431542 (0 hour treatment time for SB 431542), data indicating that the proportion of CD34+ CD 43-cells in EB was significantly increased after SB431542 treatment, and was optimized at 24 hours treatment time for SB 431542.

Example 10: the bFGF concentration is optimized to find that the bFGF concentration influences the proportion of CD34+ KDR + cells in EB on the fifth day

When EB differentiation was performed using T25 flasks, other conditions were performed as described above, gradient optimization of bFGF concentration was performed by treating cells with 0ng/mL, 5ng/mL, 10ng/mL, or 20ng/mL bFGF at D1-D5, respectively, and measuring CD34 and KDR expression at D5 using flow cytometry.

The results are shown in FIG. 10. The effect of bFGF concentration on differentiation efficiency was seen to present a bell-shaped curve. When the bFGF concentration is 0, the differentiation efficiency is close to 0, and when the bFGF concentration is 20ng/mL, the differentiation efficiency is decreased. The preferred concentration of bFGF is 10 ng/mL.

Example 11: CHIR99021 and SB431542 treatment significantly increased the proportion of KDR + CD 73-cells in the CD34+ CD 43-population

When EB differentiation was performed using T25 flasks, other conditions were as described above, D1-D3 treated cells with 6uM CHIR99021 and D3-D4 treated cells with 6uM SB431542, and the expression of CD34, CD43, KDR and CD73 was determined at D5 using flow cytometry, as compared to conditions without CHIR99021 and SB431542 treatments.

The results are shown in FIG. 11. Combined treatment with CHIR99021 and SB431542 significantly increased the proportion of CD34+ CD 43-cells (by about 7-fold) compared to treatment without CHIR99021 and SB 431542.

Meanwhile, more than 97% of the resulting CD34+ CD 43-cells were KDR-without treatment with CHIR99021 and SB431542 and only 57% thereof was CD73-, whereas more than 80% of the resulting CD34+ CD 43-cells were KDR + and more than 84% were CD 73-with combined treatment with addition of CHIR99021 and SB431542, showing that a very homogeneous CD34+ CD43-KDR + CD 73-population was obtained.

Example 12: the capability of the hematopoietic progenitor cells obtained by combined treatment and differentiation of CHIR99021 and SB431542 to differentiate into CD43+ CD45+ blood precursor cells is greatly improved

CD43 and CD45 are surface markers of mature blood precursor cells. When EB differentiation was performed using T25 flasks, other conditions were as described above, D1-D3 treated cells with 6uM CHIR99021 and D3-D4 treated cells with 6uM SB431542, and compared with the conditions without CHIR99021 and SB431542 treatments. To verify the ability of the hematopoietic progenitor cells obtained to differentiate further into blood precursor cells, D5EB obtained as described above was seeded on OP9 trophoblast cells and the ratio of CD43 to CD45 was measured in suspension cells after 7 days of continued culture.

The results are shown in FIG. 12. The results show that the ratio of the hematopoietic progenitor cells obtained by the treatment of CHIR99021 and SB431542 to differentiate into CD43+ CD45+ double positive cells is increased from 28.99% to 90.68%, and the increase is over 3 times, which indicates that the capability of the hematopoietic progenitor cells obtained by the combined treatment of CHIR99021 and SB431542 to differentiate into blood precursor cells is obviously improved.

Example 13: CD34+ CD43-KDR + CD 73-cells have the ability to differentiate into various blood cells

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

When EB differentiation was carried out using T25 flask, other conditions were as described above, and the experiments of blood cell clone formation were carried out by treating cells with 6uM CHIR99021 for D1-D3 and 6uM SB431542 for D3-D4, and digesting the resulting EBs of D6, D7, D8, and D9 into single cells.

The results are shown in FIG. 13. The results showed that the hematopoietic progenitor cells obtained by the above method on different days, such as D6, D7, D8, and D9, all had the ability to form a number of representative blood precursor cell clones, indicating the stability of the method.

All documents referred to herein are incorporated by reference into this application as if each were individually incorporated by reference. Furthermore, it should be understood that various changes and modifications of the present invention can be made by those skilled in the art after reading the above teachings of the present invention, and these equivalents also fall within the scope of the present invention as defined by the appended claims.

Claims

1. A hematopoietic progenitor cell differentiated from a pluripotent stem cell and having the phenotype CD34+ KDR + CD43-CD 73.

2. The hematopoietic progenitor cell of claim 1, wherein the hematopoietic progenitor cell has the phenotype CD34+ KDR + CD43-CD73-DLL4 +;

preferably, the hematopoietic progenitor cells have the phenotype CD34+ KDR + CD43-CD73-DLL4+ CD 184.

3. The hematopoietic progenitor cell of claim 1, wherein the hematopoietic progenitor cell is a human hematopoietic progenitor cell;

preferably, the pluripotent stem cell is a human pluripotent stem cell, including a human Embryonic Stem Cell (ESC) and a human Induced Pluripotent Stem Cell (iPSC).

4. The hematopoietic progenitor cell of claim 1, wherein the hematopoietic progenitor cell has any one or more characteristics selected from the group consisting of (a):

(i) more than 90% of the cells have the surface antigen CD 34;

5. The hematopoietic progenitor cell of claim 4, wherein the hematopoietic progenitor cell has 3, 4, 5, or 6 or more characteristics of group (A).

6. The hematopoietic progenitor cell of claim 1, which has the ability to differentiate into CD43+ CD45+ blood precursor cells; and/or

Said hematopoietic progenitor cells having the ability to differentiate into erythroid blood cells; and/or

Said hematopoietic progenitor cells having the ability to differentiate into myeloid lineage blood cells; and/or

The hematopoietic progenitor cells also have the ability to differentiate into lymphocytes.

7. A pharmaceutical composition for treating hematological disorders, said pharmaceutical composition comprising: an effective amount of the hematopoietic progenitor cells of claim 1, and a pharmaceutically acceptable carrier.

8. A method for serum-free preparation of hematopoietic progenitor cells comprising the steps of:

(a) providing a pluripotent stem cell;

(d) (ii) inducing culture of said mesoderm in the presence of a compound TGF- β inhibitor, thereby forming hematogenic endothelial cells; and

(e) transforming and culturing said hematopoietic endothelial cells in the presence of a combination of blood cell growth factors to obtain hematopoietic progenitor cells.

9. The method of claim 8, wherein in step (b) the culture is performed in a maintenance medium for CD34A or pluripotent stem cells supplemented with a ROCK inhibitor or other compound that promotes the survival of single human pluripotent cells.

10. Use of a hematopoietic progenitor cell according to claim 1 for the preparation of a medicament for the treatment of a hematological disorder.