CN111607566B

CN111607566B - Method for differentiating human pluripotent stem cells into hematopoietic progenitor cells and application thereof

Info

Publication number: CN111607566B
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: 2023-04-14
Anticipated expiration: 2039-02-22
Also published as: CN111607566A

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 invention relates to the field of biotechnology, in particular to a method for differentiating human pluripotent stem cells into hematopoietic progenitor cells and application 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 rate of malignant cancers of children in China is on the rise, and data in 2014 show that the incidence rate of leukemia in 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 amount of autologous HSC stored in the cord blood bank is short-lived, limiting its clinical application 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 a strong need in the art to find a safer, lower cost, 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 ethical property, and provides possibility for clinical transplantation application 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 a method of embryoid body differentiation under the effect of exogenous addition of a mixture of BMP-4 and cytokines (Chadwick K et al, 2003).

Ledran MH et al reported a method for obtaining CD 34-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 report 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).

Uensishi GI et al found that activation of the Notch signaling pathway is critical in the process of forming mature HE (uensishi GI et al, 2015).

Although the art has understood 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, low 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+ CD184-.

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 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 the cells have the surface antigen combination CD34+ CD184-.

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, dextrose, water, DMSO, and combinations thereof.

In another preferred embodiment, the concentration of said hematopoietic progenitor cells is 1X 10 ³ 1X 10 pieces/ml ⁷ One/ml, preferably 1X 10 ⁴ -1×10 ⁶ One/ml, more preferably 1X 10 ⁵ Per ml-9.9X 10 ⁵ One 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 method for serum-free preparation of 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) 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 said hematopoietic progenitor cells of 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 0.1X 10 ⁶ -5× 10 ⁶ /ml。

In another preferred embodiment, in step (b), the cells are cultured in a CD34A or pluripotent stem cell maintenance medium supplemented with a ROCK inhibitor or other compound that promotes the survival of individual 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, narcisplaine or combinations thereof.

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

In another preferred example, in step (b), the suspension culture is performed 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 CHIR99021.

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

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

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 0-50ng/mL; the bFGF concentration is 0-50ng/mL; and VEGF concentration is 1-100ng/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, L Y573636, 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 may be 0.1 to 10uM, 0.3 to 10uM,0.5 to 10uM, or 1 to 10uM.

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 to 50ng/mL; the bFGF concentration is 0-20 ng/mL; and VEGF concentration is 1-100ng/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, IL7; 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 of 10-500ng/mL, preferably concentration of 100ng/mL;

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

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

the concentration of IL3 is 5-300ng/mL, and the preferable concentration can be 50ng/mL;

the concentration of IL6 is 5-300ng/mL, and the preferable concentration can be 50ng/mL;

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

the concentration of IL11 is 5-200ng/mL, and the preferable concentration can be 10ng/mL;

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

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

In another preferred embodiment, in 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 repeated herein, depending on the 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 bodies into 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 a cell phenotype in one embodiment of the present invention.

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

FIG. 7 (7A-7B) shows the identification of further refinements of hematopoietic progenitor cell phenotype in one embodiment of the present invention.

FIG. 8 (8A-8C) shows a comparative experiment with the known method during the optimization of the technique of the present invention. Compared with the known method without CHIR99021 (concentration of CHIR99021 is 0), the treatment of CHIR99021 obviously 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 obviously improved after the treatment of CHIR99021, and the optimal proportion is reached when the concentration of CHIR99021 is 6uM.

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 progenitor cells compared to the method without SB431542 (0 hour treatment time for SB 431542), data indicating that the proportion of CD34+ CD 43-cells in EBs was significantly increased after SB431542 treatment, and was optimal at 24 hours treatment time for SB431542.

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, making the hematopoietic progenitor cell phenotype obtained by differentiation more uniform. In the figure, CHIR denotes CHIR99021, and SB denotes SB431542.

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 SB431542.

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 hematopoietic progenitor cells having a stable differentiation effect can be efficiently and rapidly obtained in the absence of serum by optimizing conditions in the presence of a specific compound, and the hematopoietic progenitor cells have a differentiation ability of both erythroid, myeloid and lymphoid lineages. The present invention has been completed on the basis of 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", usually 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 a less specialized cell forms 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 cords 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 "CHIR99021" is intended to mean that the compound having a CAS No. 252917-06-9, and the term also includes CHIR99021 and salts, especially pharmaceutically acceptable salts thereof. The chemical structure of CHIR99021 is:

as used herein, the term "SB431542" 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 a specific serum-free condition based on a specific small molecular compound.

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, thus removing the effects of primitive hematopoiesis; (3) The final product prepared by the method has the cell phenotype of CD34+ KDR + CD43-CD73-, and is mature hematopoietic progenitor cells with high T cell differentiation capacity.

In the present invention, in addition to the specific small molecule compounds CHIR99021 and SB431542 used in different culturing stages, conditions such as the concentration of the small molecule compounds and the culturing time are optimized, so that the optimized method of the present invention can rapidly and efficiently prepare hematopoietic progenitor cells, and the prepared hematopoietic progenitor cells have the ability to stably differentiate into various blood cells (including cells having erythroid, myeloid and lymphoid lineages).

It will be appreciated that in the method of the invention, in addition to using specific small molecule compounds CHIR99021 and SB431542 at different stages of cultivation, culture media and cultivation 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 added to the medium or culture system.

In the present invention, representative additional GSK3beta inhibitors include: BIO (CAS No. 667463-62-9), TWS119 (CAS No. 601514-19-6), kenpaulolone (CAS No. 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 No. 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 induction of 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: prior to the delivery or use of each batch of hematopoietic progenitor cells, they must be checked for sterility, endotoxin and mycoplasma, and DNA identity. The cells distributed in each batch are required to meet the cell activity of 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 result of acute toxicity and allergy detection of the hematopoietic progenitor cells is 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 after purification and enrichment is preferably greater than or equal to 90%.

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 a leukocyte sialoprotein 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 are labeled using the CD 43-negative characteristic. In the present invention, the proportion of CD 43-negative hematopoietic progenitor cells in the total cell population after purification and enrichment is preferably greater than or equal to 90%.

The CD73 antigen is an extracellular-5' nucleotidase and is also a signal transduction molecule expressed on several subpopulations of T and 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 CD73 negative characteristic. In the present invention, the proportion of CD 73-negative hematopoietic progenitor cells in the total cell population after purification and enrichment is preferably greater than or equal to 90%.

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 dysfunction of 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 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 not less than 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 taken 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 70% or more.

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) ₂ A fragment; single chain Fv molecules (scFV); or a chimeric antibody. Antibodies are added to bind to antigens 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 is functional or active and acceptable to 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 response), i.e., at a reasonable benefit/risk ratio. The term "pharmaceutically acceptable carrier" refers to a carrier, including various excipients and diluents, for administration of a therapeutic agent.

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 composition of the present invention may 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 immunological status of the patient, the route of administration, etc.

The pharmaceutical composition of the present invention is preferably an intravenous agent. In another preferred embodiment, the concentration of the hematopoietic progenitor cells in the intravenous injection is 1 × 10 ³ 1X 10 pieces/ml ⁷ One/ml, preferably 1X 10 ⁴ -1×10 ⁶ One/ml, more preferably 1X 10 ⁵ Per ml-9.9X 10 ⁵ Each/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 aplasia, multiple myeloma.

The main advantages of the invention include:

firstly, the hematopoietic progenitor cells obtained by the method have the ability 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 percent; wherein the phenotype CD34+ KDR + CD43-CD 73-cells account for more than 80% of the phenotype CD34+ cells and have strong capacity of differentiating into T cells;

thirdly, the whole differentiation process uses a serum-free culture medium without using trophoblast cells, and is suitable for the production of clinical 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 is as follows:

serial number	Composition (A)	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, etc. can be added to the basic 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 degradation of erythrocytes. The transferrin is 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 the molecular formula 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), 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, are Basal cell culture media such as Iscove's modified Dulbecco's Medium (IMDM liquid Medium), 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 90% degree of polymerization in good undifferentiated state were digested into single cell suspension, suspended in human pluripotent stem cell medium at a certain density, and cultured on a shaker in an incubator at 37 ℃ 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 iPSC is normally cultured in an iPSC maintenance medium, and the used medium is E8 or TeSR or other similar mediums.

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 enzyme digests ipscs into a complete single cell suspension, and the dispersed pluripotent cells are seeded into CD34A or pluripotent stem cell maintenance medium at a density of about 0.1 million to about 5 million/mL. 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 incubated in a 37 ℃ incubator by shaking, rotating or stirring or other non-static means, such as a high volume 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 the average diameter, the median or the 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, a T25 flask was used, and the concentration of Blebbistatin was 2.5uM; 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 of D1 was resuspended in fresh CD34A medium supplemented with BMP-4, bFGF, and VEGF, and GSK3 β inhibitor CHIR99021 was added to the medium at an optimized concentration, and shaking culture was continued on a shaker in an incubator at 37 ℃ for 2 days, and the EB volume continued to increase, resulting in more uniform mesodermal differentiation. The experimental method is as follows:

the flask containing the D1EB (example 1) 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, as well as the cytokines BMP-4, bFGF, VEGF, and the small molecule compound CHIR99021.

In this example, a T25 flask was used for the culture, in which BMP-4 was at a concentration of 1ng/mL; the concentration of bFGF is 0.5ng/mL; the concentration of VEGF was 5ng/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 TGF-beta inhibitor SB431542 was added to the medium and continued to be cultured on a shaker in a 37 ℃ incubator for 1 day with continued increase in EB volume, inducing mesodermal cells to differentiate 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 SB431542.

In this example, a T25 flask was used for the culture, in which the concentration of BMP-4 was 0.5ng/mL; the concentration of bFGF is 1ng/mL; the concentration of VEGF was 5ng/mL. The concentration of SB431542 was 6uM. 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 optimal concentrations of combinations of blood cell growth factors (including but not limited to SCF, TPO, FLT3L, IL3, IL6, IGF1, IL 11) were added to the media, and culturing was continued for 1-6 days on a shaker in a 37 ℃ incubator with shaking to induce EHT process to obtain CD34 positive hematopoietic progenitor cells.

The experimental method is as follows:

the flask containing the D4EB 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 bFGF, VEGF, and blood cell growth factor combination. In this example, the bFGF concentration is 2ng/mL; VEGF concentration was 5ng/mL. There are 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 + CD73-; wherein the proportion of CD34+ cells to total cell number reached 33.21% (fig. 5A); the proportion of CD 43-cells to CD34+ cells reached 91.75% (fig. 5B), thus the proportion of CD34+ CD 43-cells to total cell number was 33.21% x 91.75% =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 the total cell number was 30.47% x 97.13% =29.60%; the proportion of CD 73-cells to CD34+ CD43-KDR + cells was 88.21% (fig. 5D), and thus the proportion of CD34+ CD43-KDR + CD 73-cells to the total cell number was 29.60% x 88.21% =26.11%. In conclusion, the proportion of 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 examination 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 proportion of CD34+ cells in the CD 34-enriched hematopoietic progenitor 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 the 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 the CD 34-enriched hematopoietic progenitor cells was 71.92% (FIG. 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: 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 performed as described above, gradient optimization of concentration of CHIR99021, i.e.cells were treated with 0uM, 6uM or 8uM of CHIR99021 at D1-D3, respectively, and the expression of KDR and CD34 was determined using flow cytometry at D5.

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

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 conditions were optimized as described above for a gradient of SB431542 treatment times, 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 measured 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 increased the efficiency of differentiation of pluripotent stem cells into hematopoietic progenitor cells compared to controls without SB431542 (0 hour treatment time for SB 431542), data indicating that the proportion of CD34+ CD 43-cells in EBs was significantly increased after SB431542 treatment, and was optimal at 24 hours treatment time for SB431542.

Example 10: the bFGF concentration is optimized, and 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 optimized as described above, using a gradient of bFGF concentration, i.e., 0ng/mL, 5ng/mL, 10ng/mL, or 20ng/mL bFGF treatment at D1-D5, respectively, and flow cytometry for CD34 and KDR expression at D5.

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 preferable concentration of bFGF is 10ng/mL.

Example 11: treatment with CHIR99021 and SB431542 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 CD34, CD43, KDR and CD73 expression was measured 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 SB431542.

Meanwhile, when more than 97% of the obtained CD34+ CD 43-cells were KDR-and only 57% thereof was CD 73-without treatment with CHIR99021 and SB431542, and when the treatment with the combination of addition of CHIR99021 and SB431542 was used, more than 80% of the obtained CD34+ CD 43-cells were KDR +, and more than 84% thereof were CD73-, it was revealed that a very uniform population of CD34+ CD43-KDR + CD 73-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, and D1-D3 cells were treated with 6uM CHIR99021 and D3-D4 cells were treated with 6uM SB431542, compared to 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 in the suspension cells was measured after 7 days of culture.

The results are shown in FIG. 12. The results show that the ratio of differentiating the hematopoietic progenitor cells obtained by treating CHIR99021 and SB431542 into CD43+ CD45+ double positive cells is increased from 28.99% to 90.68%, and is increased by more than 3 times, which shows that the capability of differentiating the hematopoietic progenitor cells obtained by treating CHIR99021 and SB431542 into blood precursor cells is obviously improved.

Example 13: CD34+ CD43-KDR + CD 73-cells have the ability to differentiate into a variety of 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 performed using T25 flasks, other conditions were as described above, and the cells were treated with 6uM CHIR99021 for D1-D3 and 6uM SB431542 for D3-D4, and the resulting EBs of D6, D7, D8 and D9 were digested into single cells to perform a blood cell clone formation experiment.

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 had been individually incorporated by reference. Furthermore, it should be understood that various changes or 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 appended claims.

Claims

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

(a) Providing a pluripotent stem cell;

(c) Inducing culture of said embryoid bodies in the presence of the compound GSK-3 β inhibitor, BMP-4, bFGF and VEGF, thereby forming mesoderm;

(d) Inducing culture of said mesoderm in the presence of the compounds TGF-beta inhibitor, BMP-4, bFGF and VEGF, thereby forming hematogenic endothelial cells; and

(e) Transforming said hematopoietic endothelial cells in culture in the presence of a combination of blood cell growth factors comprising SCF and TPO, thereby obtaining hematopoietic progenitor cells;

wherein the GSK-3 beta inhibitor is one of the following compounds or the combination thereof: NP031112, TWS119, SB216763, CHIR-98014, AZD2858, AZD1080, SB415286, LY2090314, and CHIR99021;

the TGF-beta inhibitor is one of the following compounds or the 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 of claim 1, wherein in step (a), comprising: subjecting the pluripotent stem cells to a digestion process, thereby forming a single cell suspension.

3. The method of claim 2, wherein in step (a), the treatment is performed with an accutase digestive enzyme.

4. The method of claim 1, wherein in step (b), the pluripotent stem cells are seeded at a concentration of 0.1 x 10 ⁶ -5×10 ⁶ /mL。

5. The method of claim 1, wherein in step (b), the culturing is effected in CD34A supplemented with a ROCK inhibitor, the composition of the medium for CD34A being as follows: a basic culture medium, transferrin with a final concentration of 2 to 200 mu g/mL, MTG with a final concentration of 100 to 400 mu M, vitamin C with a final concentration of 20 to 100 mu g/mL, glutamine with a final concentration of 0.5 to 5mM and recombinant human insulin with a final concentration of 0.5 to 5 mu g/mL.

6. The method of claim 5, wherein the ROCK inhibitor is selected from the group consisting of: blebbistatin, HA-100, Y-27632, HA-1077, KD-025, Y-33075, narcilase or a combination thereof.

7. The method of claim 5, wherein in step (b), the ROCK inhibitor is Blebbistatin.

8. The method of claim 5, wherein in step (b), the suspension culture is carried out for a period of 4 to 32 hours.

9. The method of claim 1, wherein the concentration of GSK-3 β inhibitor is 0.1 to 20uM.

10. The method of claim 1, wherein in step (c), the culturing is for a period of 1 to 3.5 days.

11. The method of claim 1, wherein in step (c), the BMP-4 concentration is 1-50ng/mL; the bFGF concentration is 0.5-50ng/mL; and VEGF concentration is 1-100ng/mL.

12. The method of claim 1, wherein in step (d) the concentration of the TGF- β inhibitor is 0.1 to 10uM.

13. The method of claim 1, wherein in step (d), the culturing is for a period of 0.5 to 4 days.

14. The method of claim 1, wherein in step (d), the concentration of BMP-4 is 0.5 to 50ng/mL; the bFGF concentration is 1-20ng/mL; and VEGF concentration is 1-100ng/mL.

15. The method of claim 1, wherein in step (e), bFGF and VEGF are also present in the culture system.

16. The method of claim 1, wherein in step (e), the culture system is free of BMP-4.

17. The method of 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 blood cell growth factor is present at a concentration of:

the concentration of SCF is 10-500ng/mL;

the concentration of TPO 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;

IL15 concentration is 5-200ng/mL.

20. The method of claim 1, wherein in steps (c), (d) and (e), said culturing is suspension culturing.

21. The method of claim 1, wherein the method further comprises: (f) And (4) detecting the cell phenotype of the formed hematopoietic progenitor cells.

22. The method of claim 1, wherein the method is a method for producing hematopoietic progenitor cells under serum-free conditions.

23. A population of hematopoietic progenitor cells prepared by the method of any one of claims 1 to 22.

24. The population of hematopoietic progenitor cells of claim 23, wherein the population is differentiated from pluripotent stem cells to provide a proportion of CD34+ hematopoietic progenitor cells greater than 30%, and wherein the cells having the phenotype CD34+ KDR + CD43-CD73 comprise greater than 80% of the CD34+ hematopoietic progenitor cells.

25. The population of hematopoietic progenitor cells of claim 24, wherein the cells having the phenotype CD34+ KDR + CD43-CD73 have the phenotype CD34+ KDR + CD43-CD73-DLL4+.

26. The population of hematopoietic progenitor cells of claim 24, wherein the cells having the phenotype CD34+ KDR + CD43-CD73 have the phenotype CD34+ KDR + CD43-CD73-DLL4+ CD184-.

27. The population of hematopoietic progenitor cells of claim 23, wherein the population of hematopoietic progenitor cells is a human hematopoietic progenitor cell population.

28. The population of hematopoietic progenitor cells of claim 24, wherein the pluripotent stem cells are human pluripotent stem cells comprising human Embryonic Stem Cells (ESC) and human Induced Pluripotent Stem Cells (iPSC).

29. The population of hematopoietic progenitor cells of claim 23, wherein the population of hematopoietic progenitor cells has any one or more characteristics selected from the group consisting of:

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

30. The population of hematopoietic progenitor cells of claim 29, wherein the population has 3, 4, 5, or 6 or more of the above characteristics.

31. The population of hematopoietic progenitor cells of claim 23, which have the ability to differentiate into CD43+ CD45+ blood precursor cells; and/or

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

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

The hematopoietic progenitor cell population also has the ability to differentiate into lymphocytes.

32. A pharmaceutical composition for treating hematological disorders, said pharmaceutical composition comprising: an effective amount of the population of hematopoietic progenitor cells of claim 23, and a pharmaceutically acceptable carrier.

33. The pharmaceutical composition of claim 32, wherein the pharmaceutical composition is a liquid formulation.

34. The pharmaceutical composition of claim 33, wherein said hematopoietic progenitor cells are present in said pharmaceutical composition at a concentration of 1 x 10 ⁴ 1X 10 per mL ⁶ one/mL.

35. The pharmaceutical composition of claim 34, wherein the hematopoietic progenitor cell is at a concentration of 1 x 10 ⁵ Per mL-9.9X 10 ⁵ One per mL.

36. Use of a population of hematopoietic progenitor cells of claim 23 for the preparation of a medicament for treating a hematological disorder selected from the group consisting of: anemia, leukemia, or a combination thereof.