WO2024060485A1

WO2024060485A1 - Method for preparing hematopoietic stem cell or hematopoietic stem and progenitor cell and method for culturing long-term hematopoietic stem cell

Info

Publication number: WO2024060485A1
Application number: PCT/CN2023/074760
Authority: WO
Inventors: 杜如龙; 于蕾; 黄雯静; 武雪宁; 顾雨春; 吴理达
Original assignee: 呈诺再生医学科技(北京)有限公司
Priority date: 2022-09-21
Filing date: 2023-02-07
Publication date: 2024-03-28
Also published as: CN115247152B; CN115247152A

Abstract

Provided herein are a method for preparing a hematopoietic stem cell or a hematopoietic stem and progenitor cell, and a method for culturing a long-term hematopoietic stem cell. The method for preparing a hematopoietic stem cell or a hematopoietic stem and progenitor cell comprises: 1) providing a hematopoietic mesoderm cell or a cell culture comprising the hematopoietic mesoderm cell; 2) placing the hematopoietic mesoderm cell or the cell culture comprising the hematopoietic mesoderm cell in a hemogenic endothelium specialization and endothelial-to-hematopoietic transition culture medium for culturing; and 3) enabling the hematopoietic mesoderm cell to express or overexpress a transcription factor OCT4. Also provided herein is a method for culturing a long-term hematopoietic stem cell or culturing a cell culture comprising a long-term hematopoietic stem cell in a culture medium, which comprises enabling the long-term hematopoietic stem cell to express a cytokine OCT4.

Description

Methods for preparing hematopoietic stem cells or hematopoietic stem progenitor cells and methods for culturing long-term regenerative hematopoietic stem cells

Technical field

This article relates to the field of cell technology, specifically methods for preparing hematopoietic stem cells or hematopoietic stem progenitor cells and culturing long-term regenerative hematopoietic stem cells.

Background technique

Hematopoietic Stem Cells (HSC) are a type of adult stem cells with self-renewal and differentiation potential, which can differentiate into all blood cells and platelets. Hematopoietic stem cells can be used to treat related blood diseases, including leukemia and lymphoma, through cell transplantation; at the same time, they can differentiate into various types of blood cells and platelets in vitro for clinical treatment and research. At present, hematopoietic stem cells are mainly isolated and obtained from the body. However, due to their extremely small content and the inability to be cultured for a long time in vitro, the clinical research and application of hematopoietic stem cells have been seriously restricted. Pluripotent Stem Cells (PSC) are a type of cells with self-renewal and differentiation potential, including embryonic stem cells (Embryonic Stem Cells (ESC)), induced pluripotent stem cells (iPSC) and expanded pluripotent stem cells (Extended Pluripotent Stem Cells, EPSC) and totipotent stem cells (Totipotent Stem Cells, TPSC), etc.; they can be induced to differentiate into hematopoietic stem cells, providing new options and approaches for the transplantation and clinical application of hematopoietic stem cells. However, the current induced differentiation method can only obtain hematopoietic progenitor cells (HPC) with limited differentiation potential and unable to self-renew for a long time, but cannot obtain hematopoietic stem cells (long-term HSC) with long-term hematopoietic function, and currently Differentiation methods have many shortcomings, including low induction efficiency, long differentiation cycle, complex differentiation process, and animal-derived components in the differentiation medium. These shortcomings seriously limit the clinical research and application of hematopoietic stem cells.

Contents of the invention

In one aspect, this article provides methods for preparing hematopoietic stem cells or hematopoietic stem progenitor cells, including:

1) Provide hematopoietic mesoderm cells or cell culture including hematopoietic mesoderm cells;

2) culturing the hematopoietic mesoderm cells or the cell culture comprising the hematopoietic mesoderm cells in a hematopoietic endothelial specialization and endothelial-hematopoietic transition medium; and

3) Let the hematopoietic mesoderm cells express the transcription factor OCT4.

In some embodiments, step 3) is allowing the hematopoietic mesoderm cells to overexpress the transcription factor OCT4.

In some embodiments, step 3) is performed 3 days after step 2).

In some embodiments, step 3) is performed for at least 4 days.

In some embodiments, step 3) is performed on days 4-7, days 4-10, or days 7-10 of culture in step 2).

In some embodiments, the hematopoietic endothelial specialization and endothelial-to-hematopoietic switching medium contains VEGF, bFGF, SCF, IL-3, TPO, Flt-3L, and BMP4;

In some embodiments, the hematopoietic endothelial specialization and endothelial-hematopoietic switching medium is STEMdiff ^™ APEL ^™ 2 medium supplemented with VEGF, bFGF, SCF, IL-3, TPO, Flt-3L and BMP4.

In some embodiments, the hematopoietic mesoderm cells comprise exogenously introduced nucleic acid sequences encoding the transcription factor OCT4.

In some embodiments, the coding nucleic acid sequence is operably linked to an inducible promoter;

In some embodiments, the inducible promoter is a tetracycline-inducible promoter;

In some embodiments, the hematopoietic mesoderm cells further comprise exogenously introduced rtTA encoding nucleic acid sequences.

In some embodiments, the coding nucleic acid sequence is integrated into the genome of the hematopoietic mesoderm cell.

In some embodiments, step 3) causes the hematopoietic mesoderm cells to express the transcription factor OCT4 by adding tetracycline or doxycycline to the hematopoietic endothelial specialization and endothelial-hematopoietic conversion medium.

In some embodiments, the hematopoietic mesoderm cells or a cell culture comprising hematopoietic mesoderm cells are obtained by culturing the mesoderm cells or a cell culture comprising mesoderm cells in a hematopoietic mesoderm-specific medium.

In some embodiments, the hematopoietic mesoderm cells or the cell culture comprising mesoderm cells are cultured in the hematopoietic mesoderm specialized medium for 2 days to obtain the hematopoietic mesoderm cells or the hematopoietic mesoderm cells. of cell cultures.

In some embodiments, the hematopoietic mesoderm-specific medium contains VEGF and bFGF.

In some embodiments, the hematopoietic mesoderm specialized medium is STEMdiff ^™ APEL ^™ 2 medium supplemented with VEGF and bFGF.

In some embodiments, the mesodermal cells or cell culture comprising mesodermal cells are obtained by mesodermal induction of pluripotent stem cells (PSC); preferably, the pluripotent stem cells are induced pluripotent stem cells (PSCs). iPSC); more preferably, the pluripotent stem cells are human induced pluripotent stem cells (hiPSC).

In some embodiments, the pluripotent stem cells comprise exogenously introduced nucleic acid sequences encoding the transcription factor OCT4. Preferably, said introduction is carried out via lentiviral vectors.

In some embodiments, the hematopoietic mesodermal cells are KDR ⁺ and PDGFRα ⁻ .

In some embodiments, the hematopoietic stem cells are CD34 ⁺ CD45RA ^- CD90 ⁺ EPCR ⁺ .

In some embodiments, the hematopoietic stem cells are long-term regenerative hematopoietic stem cells.

In some embodiments, the long-term regenerative hematopoietic stem cells are CD34 ⁺ EPCR ⁺ CD90 ⁺ ITGA3 ⁺ .

In some embodiments, the hematopoietic stem and progenitor cells are CD34 ⁺ and CD45 ⁺ .

In some embodiments, the mesodermal cells are Braychury ⁺ .

In another aspect, provided herein are methods of culturing long-term regenerative hematopoietic stem cells or culturing a cell culture comprising long-term regenerative hematopoietic stem cells in a culture medium, comprising allowing the long-term regenerative hematopoietic stem cells to express or overexpress the cytokine OCT4.

In some embodiments, the expression or overexpression of the cytokine OCT4 is performed on days 1-3 of culturing the long-term regenerative hematopoietic stem cells or a cell culture comprising long-term regenerative hematopoietic stem cells.

In some embodiments, the expression or overexpression of the cytokine OCT4 is performed in one of the following ways:

1) Adding a cytokine OCT4 expression activator to the culture medium; and

2) Introducing the exogenous nucleic acid sequence encoding the transcription factor OCT4 into the long-term regenerative hematopoietic stem cells or their precursor cells.

In some embodiments, the activator of cytokine OCT4 expression is OAC1.

In some embodiments, the nucleic acid sequence encoding the transcription factor OCT4 is operably linked to an inducible promoter.

In some embodiments, the inducible promoter is a tetracycline-inducible promoter

In some embodiments, the long-term regenerative hematopoietic stem cells or precursor cells thereof further include exogenously introduced rtTA encoding nucleic acid sequences.

Beneficial Effects

The present invention establishes a serum-free differentiation system and an easy-to-operate differentiation process, which regulates the key signaling pathways related to the development of hematopoietic stem cells through stage-specificity and induces the development of hematopoietic stem cells through the Tet-on tetracycline inducible expression system. The expression of the process-related core transcription factor OCT4 achieves efficient differentiation of human pluripotent stem cells into CD34 ⁺ EPCR ⁺ CD90 ⁺ ITGA3 ⁺ long-term regenerative hematopoietic stem cells in vitro. At the same time, we found that in short-term in vitro expansion culture, overexpression of OCT4 promoted the proliferation of CD34 ⁺ EPCR ⁺ CD90 ⁺ ITGA3 ⁺ long-term regenerative hematopoietic stem cells. This study expands the method for in vitro differentiation of human pluripotent stem cells into hematopoietic stem cells and may provide a new source of hematopoietic stem cells for future clinical applications and research.

Description of drawings

Figure 1 is a flow chart of a specific embodiment of differentiation of human pluripotent stem cells (hiPS-001-5-OCT4) into hematopoietic stem cells. The differentiation process of human pluripotent stem cells into hematopoietic cells mainly includes monolayer cell formation, mesoderm induction, hematopoietic mesoderm specialization, hematopoietic endothelial specialization and endothelial-hematopoietic conversion.

Day-1-0 single cells were formed using TeSR-E8 medium with a cell density of 8000 cells/cm ² and 10 μM Y-27632 added;

Day0-1 mesoderm induction medium, containing 9μM CHIR99021 (CHIR);

Day 1-3 Hematopoietic mesoderm specialized medium, containing 20ng/mL VEGF and 20ng/mL bFGF;

Day3-6 hematopoietic endothelial specialization and endothelial-hematopoietic conversion medium, containing 20ng/mL VEGF, 20ng/mL bFGF, 50ng/mL SCF, 10ng/mL IL-3, 30ng/mL TPO, 10ng/mL Flt-3L and 10ng/mL BMP4. Cells were passaged on Day 3. The cell seeding density was 2×10 ⁴ cells/cm ² . An additional 10 μM Y-27632 was added. After 24 hours, the culture medium was replaced and Y-27632 was removed.

Day6-12 hematopoietic endothelial specialization and hematopoietic endothelial-hematopoietic conversion medium, containing 20ng/mL VEGF, 20ng/mL bFGF, 50ng/mL SCF, 10ng/mL IL-3, 30ng/mL TPO, 10ng/mL Flt-3L , 10ng/mL BMP4 and 5μg/mL Doxycycline (DOX), among which DOX induces OCT4 overexpression. Thereafter, fresh DOX-containing hematopoietic endothelial specialization and endothelial-hematopoietic conversion medium was replaced every two days until Day 12.

Figure 2 Overexpression of OCT4 promotes the generation of long-term regenerative hematopoietic stem cells. (A) qRT-PCR analysis of the induction of OCT4 gene transcriptional expression in hiPS-001-5-OCT4 cell line after 3 days of DOX treatment. - represents no addition of DOX; + represents addition of DOX; DOX represents 5μg/mL doxycycline. (B) Flow cytometry analysis of the differentiation efficiency of CD34 ⁺ KDR ⁺ hematopoietic endothelial cells induced by adding 5μg/mL doxycycline to D3-6 on day 6 of differentiation. Control represents the control group without addition of DOX; D3-6 represents the addition of 5μg/mL doxycycline from days 3 to 6 of differentiation. (C) Flow cytometry analysis of the differentiation efficiency of CD34 ⁺ CD45 ⁺ hematopoietic cells induced by adding 5μg/mL doxycycline at different time windows on day 9 of differentiation. Control represents the control group without addition of DOX; D3-9 represents the addition of 5μg/mL doxycycline from days 3 to 9 of differentiation; D6-9 represents the addition of 5μg/mL doxycycline from days 6 to 9 of differentiation. (DG) Flow cytometry analysis of the differentiation efficiency of CD34 ⁺ CD90 ⁺ EPCR ⁺ ITGA3 ⁺ long-term regenerative hematopoietic stem cells induced by adding 5 μg/mL doxycycline at different time windows on day 12 of differentiation. Control represents the control group without the addition of doxycycline; D3-12 represents the addition of 5 μg/mL doxycycline on days 3 to 12 of differentiation; D6-9 represents the addition of 5 μg/mL doxycycline on days 6 to 9 of differentiation; D6-12 represents the addition of 5 μg/mL doxycycline on the 6th to 12th day of differentiation; D9-12 represents the addition of 5 μg/mL doxycycline on the 9th to 12th day of differentiation; among them, Figure D and Figure E are experiments from different batches, and the treatment time windows partially overlap, mainly to find the optimal action time window, Figure F reflects the line graph of Figure D; Figure G reflects the line graph of Figure E.

Figure 3 Overexpression of OCT4 maintains long-term regeneration of hematopoietic stem cells in vitro culture. (A) Flow cytometric analysis of the effect of adding 5 μg/mL doxycycline on the maintenance of CD34 ⁺ CD90 ⁺ EPCR ⁺ ITGA3 ⁺ long-term regenerative hematopoietic stem cells on the third day of in vitro expansion culture. D0 represents the starting cells; Control represents the control group, no DOX is added; DOX represents the addition of 5 μg/mL doxycycline. (B) After the 3rd day of in vitro expansion and culture, the cell expansion fold was analyzed, and the effect of adding 5 μg/mL doxycycline on the proportion of CD34 ⁺ CD90 ⁺ EPCR ⁺ ITGA3 ⁺ long-term regenerative hematopoietic stem cells and the absolute number of cells were analyzed by flow cytometry. D0 represents the starting cells; D3-Control represents the control group, without adding DOX; D3-DOX represents the addition of 5 μg/mL doxycycline.

Figure 4 Cell morphology diagram of human pluripotent stem cells (hiPS-001-5-OCT4) before passage (Day-1) at different magnifications. Observed under an ordinary light microscope, the cell confluence of human pluripotent stem cells (hiPS-001-5-OCT4) before passage was about 70% to 80%; the edges of the cell clones were smooth, and no obviously differentiated cells were seen. The cells were tightly arranged and had a relatively three-dimensional effect. good.

Figure 5 Cell morphology diagram of human pluripotent stem cells (hiPS-001-5-OCT4) before induced differentiation (Day0) at different magnifications. Observed under ordinary light microscope, human pluripotent stem cells (hiPS-001-5-OCT4) cells formed smaller clones before differentiation.

Figure 6 Morphological diagram of mesodermal cells induced and differentiated by human pluripotent stem cells (hiPS-001-5-OCT4) at different magnifications (Day1). Observation under an ordinary light microscope showed that human pluripotent stem cells (hiPS-001-5-OCT4) cells were induced to differentiate into mesoderm cells, and the edges of the cell clones shrank significantly after mesoderm induction.

Figure 7 Flow cytometric detection results of mesoderm markers induced and differentiated by human pluripotent stem cells (hiPS-001-5-OCT4) (Day1). Through cell flow cytometry analysis, the expression of mesodermal cell marker T (Braychury) in induced differentiation of human pluripotent stem cells (hiPS-001-5-OCT4) cells. For specific detection methods, please refer to Cell Flow Cytometry in the Experimental Methods section. Note: After mesoderm induction, cell clones should undergo edge shrinkage changes, and the induction efficiency of the mesoderm marker T (Braychury) should reach more than 90% by flow cytometry.

Figure 8 Morphological diagram of hematopoietic mesoderm cells induced and differentiated by human pluripotent stem cells (hiPS-001-5-OCT4) at different magnifications (Day3). Observed under an ordinary optical microscope, human pluripotent stem cells (hiPS-001-5-OCT4) cells are induced to differentiate into hematopoietic mesoderm cells. Morphological diagram. After induction of hematopoietic mesoderm, the cells proliferate rapidly. The cells take on a mesenchymal-like cell shape and the cells are polygonal. , the arrangement is relatively loose.

Figure 9 Flow cytometric detection results of hematopoietic mesoderm markers induced by human pluripotent stem cells (hiPS-001-5-OCT4) differentiation (Day3). Through cell flow cytometry analysis, the expression of human pluripotent stem cells (hiPS-001-5-OCT4) cells induced differentiation of mesodermal cell markers KDR and PDGFRα. For specific detection methods, please refer to Cell Flow Cytometry in the Experimental Methods section. Note: During the hematopoietic mesoderm induction stage, cells will rapidly spread and proliferate. Compared to compact clones, cells become relatively loose. The proportion of hematopoietic mesoderm KDR ⁺ PDGFRα _- cells detected by flow cytometry should be above 70%. The cell seeding density was controlled at 1×10 ⁴ cells/cm ² to 4×10 ⁴ cells/cm ² .

Figure 10 Morphological diagram of hematopoietic endothelial cells induced and differentiated by human pluripotent stem cells (hiPS-001-5-OCT4) at different magnifications (Day4). Observation under an ordinary optical microscope showed that human pluripotent stem cells (hiPS-001-5-OCT4) cells were induced to differentiate into hematopoietic endothelial cells. After the cells were re-passed, the cell density was low, and the cells were still in the form of mesenchymal cells and were polygonal.

Figure 11 Morphological diagram of hematopoietic endothelial cells induced and differentiated by human pluripotent stem cells (hiPS-001-5-OCT4) at different magnifications (Day6). Observation under an ordinary light microscope shows that human pluripotent stem cells (hiPS-001-5-OCT4) cells are induced to differentiate into hematopoietic endothelial cells morphologically. The cells proliferate rapidly and generate more hematopoietic endothelial cells. The cells are closely arranged and short spindle-shaped. Distinct nucleolus.

Figure 12 Flow flow detection results of hematopoietic endothelial cell markers induced by human pluripotent stem cells (hiPS-001-5-OCT4) differentiation (Day6). Through cell flow cytometry analysis, the expression of human pluripotent stem cells (hiPS-001-5-OCT4) cells induced differentiation of hematopoietic endothelial cell markers CD34, KDR and CD144. For specific detection methods, please refer to Cell Flow Cytometry in the Experimental Methods section. Note: In the hematopoietic endothelial stage, the cells gradually change from mesenchymal-like cells to hematopoietic endothelial-like cells. The cells are short spindle-shaped, tightly arranged, and have obvious cell nucleoli. Flow cytometry detects hematopoietic endothelial cell markers CD34, KDR and CD144. The proportion of CD34 ⁺ KDR ⁺ cells should be no less than 15%, and the proportion of CD144 ⁺ cells among CD34 ⁺ KDR ⁺ cells should be no less than 30%.

Figure 13 Morphological diagram of induced differentiation of hematopoietic stem and progenitor cells from human pluripotent stem cells (hiPS-001-5-OCT4) at different magnifications (Day9). Observation under an ordinary optical microscope shows that human pluripotent stem cells (hiPS-001-5-OCT4) cells are induced to differentiate into hematopoietic stem progenitor cells. The hematopoietic endothelial cells migrate and gather to form a hematopoietic center, and a small number of non-adherent, round hematopoietic stem cells begin to appear. Progenitor cells.

Figure 14 Flow flow detection results of hematopoietic endothelial cell markers induced by human pluripotent stem cells (hiPS-001-5-OCT4) differentiation (Day9). Through cell flow cytometry analysis, the expression of human pluripotent stem cells (hiPS-001-5-OCT4) cells induced differentiation of hematopoietic endothelial cell markers CD34, KDR and CD144.

Figure 15 shows the morphology of hematopoietic stem cells induced by human pluripotent stem cells (hiPS-001-5-OCT4) at different magnifications (Day 12). Under ordinary optical microscope observation, human pluripotent stem cells (hiPS-001-5-OCT4) cells induced differentiation of hematopoietic stem cells to form a large number of non-adherent, round hematopoietic stem and progenitor cells.

Figure 16 Flow flow detection results of hematopoietic stem cell markers induced differentiation of human pluripotent stem cells (hiPS-001-5-OCT4) (Day12). Through cell flow cytometry analysis, human pluripotent stem cells (hiPS-001-5-OCT4) cells were induced to differentiate and long-term regenerative hematopoietic stem cell markers CD34, CD90, EPCR and ITGA3 expression. For specific detection methods, please refer to Cell Flow Cytometry in the Experimental Methods section. Note: During detection, you can use a pipette to rinse the cells repeatedly to ensure that most non-adherent round cells are collected.

Figure 17 is a graph showing the results of colony-forming unit assays (CFU, Colony-Forming Unit Assays) verifying the in vitro differentiation potential of hematopoietic stem cells. In this Example 4, the hematopoietic stem cells induced and differentiated by hiPS001-5-OCT4 were cultured in methylcellulose medium for 14 days and then formed multi-lineage progenitor cells (CFU-GEMM), granulocytes (CFU-G), macrophages ( Bright field images of colony units of CFU-M), granulocytes/macrophages (CFU-GM), and erythrocytes (B/C-FUE).

Detailed ways

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.

The term "or" refers to a single element of a listed alternative element, unless the context clearly dictates otherwise.

The term "and/or" refers to any one, any two, any three, any more or all of the listed optional elements.

"Comprising" means including the stated element, integer or step but not excluding any other element, integer or step. When "comprises" is used, it also encompasses a combination of the stated elements, integers or steps unless otherwise stated.

“Stem cells” refer to undifferentiated or insufficiently differentiated cells that are capable of self-renewing, that is, producing more cells identical to themselves, and that are capable of differentiating into two or more mature Cell type. According to the source of stem cells, stem cells can be divided into embryonic stem cells (ES cells) and adult stem cells. Embryonic stem cells can be derived from early animal embryos, such as the inner cell mass of blastocysts (i.e. early embryos), and have the ability to differentiate into every cell type in the body (totipotency). Adult stem cells are found in various organs and tissues of the adult body and have the ability to differentiate and replace cells in the tissue in which they are located (pluripotency). Hematopoietic stem cells (HSC) are adult stem cells that exist in the bone marrow and have the ability to differentiate into various blood cells. Hematopoietic stem cells (HSCs) can generate both myeloid and lymphoid progenitor cells, which in turn generate myeloid cells (such as monocytes, macrophages, neutrophils, basophils, dendritic cells, and red blood cells). , platelets, etc.) and lymphoid cells (such as T cells, B cells, NK cells, etc.). The ability of stem cells to self-replicate and differentiate into multiple or specific cell types makes them central to cell replacement therapies.

"Induced pluripotent stem cells (iPSC)" refers to stem cells with totipotency or pluripotency obtained from certain adult cells (such as fibroblasts) by artificially inducing the expression of certain genes. In some methods known in the art, iPSCs can be obtained by transfecting certain stem cell-related genes into non-pluripotent cells such as adult fibroblasts. Transfection can be achieved by viral transduction using viruses such as retroviruses or lentiviruses. In some methods, transfected genes may include the transcription factors Oct4, Sox2, Klf4, and c-Myc, although simultaneous transfection of other genes may increase induction efficiency. In other methods, lentiviral systems can be used to transform somatic cells with Oct4, Sox2, Nanog and Lin28 genes. Genes whose expression is induced in iPSCs include, but are not limited to, Oct-3/4; certain members of the Sox gene family (e.g., Soxl, Sox2, Sox3, and Sox15); certain members of the Klf family (e.g., Klfl, Klf2, Klf4, and Klf5 ), some members of the Myc family (such as C-myc, L-myc and N-myc), Nanog, Lin28, Tert, Fbx15, ERas, ECAT15-1, ECAT15-2, Tcl1, β-Catenin, ECAT1, Esg1 , Dnmt3L, ECAT8, Gdf3, Fth117, Sal14, Rex1, UTF1, Stella, Stat3, Grb2, Prdm14, Nr5a1, Nr5a2 or E-cadherin, or any combination thereof. Various reagents for preparing iPSCs, such as reprogramming vectors, expression cassettes, culture media, etc., and even commercial iPSCs are currently available on the market. hiPSC refers to iPSC induced from human cells. In a specific example, the hiPSCs used are prepared according to the method described in Chinese patent publication CN113462638A (for example, using a combination of reprogramming factors OCT4, SOX2, E6, and E7), which is hereby incorporated by reference in its entirety.

"Mesodermal cells" refers to the cell layer between the ectoderm and endoderm at the end of the gastrula during the embryonic development of triplodermal animals. Mesodermal cells can develop into the dermis, muscles, bones and other connective tissues and circulatory systems of the body, including the heart, blood vessels, bone marrow, lymph nodes, lymphatic vessels, etc.; the end of the body cavity, the serosa and mesangium of the viscera, and the connective tissue in the viscera , blood vessels and smooth muscles, etc.; kidneys, urethra, gonads (excluding germ cells), reproductive ducts, adrenal cortex, etc. In this article, mesoderm cells refer to cells with mesoderm cell markers (such as Braychury) produced by induced pluripotent stem cells (iPSCs) after culture in mesoderm induction medium. Correspondingly, the process of inducing and culturing iPSCs into mesoderm cells is called "mesoderm induction." Methods for producing mesoderm cells from induced pluripotent stem cells (iPSCs) are known in the art. For example, there are commercialized mesoderm induction media, such as STEMdiff ^TM mesoderm induction media; in addition, Chinese patent publication CN 111321110 A describes Methods for inducing mesodermal cells from iPSCs. Chinese patent publication CN106867961A describes culture media and methods for inducing mesodermal cells from iPSCs. These patent documents are incorporated herein by reference. In a specific example provided herein, mesoderm cells are obtained by culturing a monolayer of adherent iPSCs in mesoderm induction medium for 1 day (approximately 24 hours). It is contemplated that the mesoderm induction phase can be longer, for example, 1.5 days, 2 days, 3 days, etc., as long as the desired mesoderm cells can be obtained. This article also provides a cell morphology diagram of hematopoietic mesoderm cells (Figure 6).

"Hematopoietic mesoderm specialization" as used herein refers to the process of inducing differentiation of mesodermal cells into "hematopoietic mesoderm cells". "Hematopoietic mesoderm cells" can be considered as the precursor cells of hematopoietic endothelial cells, and their cell markers are KDR ⁺ PDGFRα ^- . In one example of this article, mesoderm cells can be cultured in mesoderm induction medium supplemented with VEGF and bFGF (also referred to as hematopoietic mesoderm specialized medium herein) for about 2 days and cell marker expression can be detected. Hematopoietic mesodermal cells are obtained from the situation. This article also provides a cell morphology diagram of hematopoietic mesoderm cells (Figure 8).

"Hematopoietic endothelial differentiation" in this article refers to the process of inducing differentiation of hematopoietic mesoderm cells into "hemogenic endothelium cells". Currently, researchers have believed that hematopoietic stem cells in vivo are derived from hematopoietic endothelial cells (for example, see Hou S , et al. Cell Res (2020), 30, 376-392). In an example in this article, hematopoietic mesoderm cells can be continued to be added with VEGF, bFGF, SCF, IL-3, TPO, Flt-3L and BMP4. Mesodermal induction medium (when used to prepare hematopoietic endothelial cells, this article is also called hematopoietic endothelial specialization medium; when used to prepare hematopoietic stem cells, this article is also called hematopoietic endothelial specialization and endothelial-hematopoietic transition Culture medium) for several days (for example, 3 to 12 days or more, such as 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 days, etc.) to obtain hematopoietic endothelial cells. The markers CD34, KDR and CD144 can be used to isolate or identify hematopoietic endothelial cells. This article also provides cell morphology diagrams of hematopoietic endothelial cells (Figure 10 and Figure 11)

"Endothelial-hematopoietic transition" as used herein refers to the process of transformation of hematopoietic endothelial cells into hematopoietic stem cells or hematopoietic stem progenitor cells. The process can ultimately generate hematopoietic stem cells with therapeutic applications, including long-term regenerating hematopoietic stem cells (LT-HSC). Hematopoietic stem cells or hematopoietic stem progenitor cells can be isolated or identified through cell markers such as CD34, CD45, CD90, CD45RA, EPCR or ITGA3. For example, in a specific example, this article identifies long-term regenerative hematopoietic stem cells through markers CD34, CD90, EPCR and ITGA3. In some embodiments, short-term regenerative hematopoietic stem cells (ST-HSC) are characterized by CD34 ⁺ EPCR ⁺ CD90 ⁺ ^ITGA3- , which can be maintained in mice for 3-6 months. In some embodiments, long-term regenerating hematopoietic stem cells (LT-HSC) are characterized by CD34 ⁺ EPCR ⁺ CD90 ⁺ ITGA3 ⁺ . ITGA3 is a functional marker of human long-term regenerative hematopoietic stem cells expanded in vitro. For example, Tomellini et al. experimentally demonstrated that ITGA3 is necessary to maintain long-term stem cell activity in vivo. ITGA3 expression is functionally required for long-term engraftment of cord blood (CB) cells. ITGA3 is a reliable marker for HSCs cultured in CB samples, improving It improved the accuracy of expected HSC identification and was able to distinguish ST-HSC and multipotent LT-HSC in expanded CB culture (Tomellini, et al. Integrin-α3Is a Functional Marker of Ex Vivo Expanded Human Long-Term Hematopoietic Stem Cells. Cell Rep. 2019;28(4):1063-1073).

"Promoter" is a DNA sequence that RNA polymerase recognizes, binds and starts transcription. It contains conserved sequences required for specific binding of RNA polymerase and initiation of transcription. Most of them are located upstream of the transcription start point of structural genes. The promoter itself Not transcribed. Examples of promoters include, but are not limited to, CMV, EF1A, CAG, CBh, SFFV promoters.

"Inducible promoter" means that, in addition to the promoter sequence, it also includes at least one transcriptional control sequence, which can initiate or promote the transcription of its downstream DNA sequence by the promoter after a specific transcription factor binds to the transcriptional control sequence. The transcription control sequence and the promoter may or may not naturally exist in the transcription control sequence of the same gene, and may be called natural inducible promoters or artificial inducible promoters respectively. In the specific examples provided herein, tetracycline-inducible promoters (artificial inducible promoters) are used, which include, for example, CMV promoter (minimal CMV promotor, PminCMV) and Tet-responsive element (Tet-responsive element, TRE). In the presence of DOX (Doxycycline), antisense Tet transcriptional activator (rtTA) binds to DOX and then binds to TRE, activating PminCMV to promote gene expression; in the absence of DOX, rtTA does not bind to TRE, and PminCMV It cannot initiate gene expression by itself. Therefore, when rtTA is expressed simultaneously in cells (either by using another expression vector, or using a host cell that has integrated the rtTA coding sequence), the activity of the inducible promoter can be controlled by whether to add DOX. Those skilled in the art can understand that in addition to tetracycline-inducible promoters, other inducible expression systems can also be used to achieve the purpose of the present invention, such as the ecdysone-inducible system, Cumate, rapamycin system and other expression systems commonly used in the field. .

"Operably linked to an inducible promoter" means that the regulatory sequence-inducible promoter is connected to its regulatory object in such a manner that the regulatory sequence-inducible promoter can exert an effect on its regulatory object. For example, a promoter operably linked to a gene of interest means that the promoter can drive transcription of the gene of interest from an accurate start site.

The term "vector" refers to a nucleic acid molecule that can be engineered to contain a polynucleotide of interest (eg, a coding sequence for a protein of interest) or a nucleic acid molecule that can replicate in a host cell (eg, nucleic acid, plasmid, or virus, etc.). The vector may include one or more of the following components: an origin of replication, one or more regulatory sequences that regulate expression of the polynucleotide of interest (such as a promoter and/or enhancer), and/or one or more Selectable marker genes (such as antibiotic resistance genes and genes useful in colorimetric assays, such as beta-galactose). The term "expression vector" refers to a vector used to express a protein of interest in a host cell. Plasmid vectors can usually be used for transient expression of target proteins in cells, while viral vectors, such as lentiviral vectors, can be used for stable expression in cells.

When referring to a nucleic acid sequence encoding a protein, "expression" refers to the transcription and/or translation of the nucleic acid sequence encoding the protein. Expression can be at a basal level, that is, the level of normal expression of a particular gene in a particular cell. Expression can also be at a super level, that is, overexpression. The amount of mRNA or protein produced is usually several times the basal level, such as 5 times, 10 times, 20 times, 50 times, 100 times, 200 times, 500 times, 1000 times or more. Introducing exogenous nucleic acid sequences (including expression cassettes encoding nucleic acid sequences encoding a protein of interest) into host cells (such as stem cells) is a way to achieve overexpression of the protein of interest in corresponding host cells. Setting a strong promoter or an inducible strong promoter in the expression cassette can further enhance the expression level.

One aspect of the present invention provides a method for preparing hematopoietic stem cells or hematopoietic stem progenitor cells, including first constructing iPSCs that can inducibly express the transcription factor OCT4, and during the induction process of the iPSCs into hematopoietic stem cells (especially the endothelial-hematopoietic conversion process) Induces the expression of this transcription factor, thereby increasing the production or yield of hematopoietic stem cells or hematopoietic stem progenitor cells (Figure 1). The inventors found that inducing the expression of this transcription factor in a specific period of time can significantly increase the proportion (or yield) of hematopoietic stem cells or hematopoietic stem progenitor cells in culture. In a specific embodiment, induction occurs on day 4 to day 7 (corresponding to Day6-Day9 in Figure 1, abbreviated as D6-9) starting from hematopoietic endothelial differentiation. Expression of the transcription factor OCT4 (i.e. addition of DOX to the culture medium). In another embodiment, expression of the transcription factor is induced on days 4 to 10 from the onset of hematopoietic endothelial differentiation (corresponding to Day6-Day12 in Figure 1). In another embodiment, expression of the transcription factor is induced on days 7 to 10 from the onset of hematopoietic endothelial differentiation (corresponding to Day9-Day12 in Figure 1). In another specific embodiment, expression of the above-mentioned transcription factors is induced starting on day 4 from the onset of hematopoietic endothelial differentiation (corresponding to Day 6 in Figure 1). The generation of hematopoietic stem cells (including hematopoietic stem progenitor cells or long-term regenerative hematopoietic stem cells) can be identified by observing cell morphology and/or detecting cell markers. In some embodiments, the method includes: 1) providing hematopoietic mesoderm cells or a cell culture comprising hematopoietic mesoderm cells; 2) placing the hematopoietic mesoderm cells or cell culture comprising hematopoietic mesoderm cells within the hematopoietic and 3) allowing the hematopoietic mesoderm cells to express the transcription factor OCT4. It should be pointed out that step 3) does not need to be performed after step 2), but is performed during the culture process of step 2).

Another aspect of the present invention is to provide a method for culturing long-term regenerative hematopoietic stem cells or a cell culture comprising long-term regenerative hematopoietic stem cells in a culture medium, which comprises allowing the long-term regenerative hematopoietic stem cells to express or overexpress the cytokine OCT4. The inventors have found that expressing the cytokine OCT4 can promote the proliferation of CD34 ⁺ EPCR ⁺ CD90 ⁺ ITGA3 ⁺ long-term regenerative hematopoietic stem cells.

Regarding the expression or overexpression of the cytokine OCT4, it is expected that those skilled in the art can use other methods to achieve similar effects to the DOX-induced expression used in the embodiments herein. These other means include, for example, introducing an OCT4 plasmid vector into the cell resulting in short-term expression of OCT4; by adding an activator of OCT4 expression, such as OAC1, etc., these modified embodiments are also included within the scope of the present invention.

Various cells mentioned in this article, such as mesodermal cells, hematopoietic mesodermal cells, hematopoietic stem cells, etc., can be identified by observing cell morphology and/or detecting cell markers. For example, cell morphology observation and marker detection can be performed on the second day of hematopoietic endothelial differentiation (corresponding to Day 4 in Figure 1). In this article, when it comes to observing cell morphology or detecting markers, the corresponding time, such as Day 4, usually refers to the beginning of that day, or it can also be before specific treatments (such as inoculation, changing culture media, etc.) of cultured cells on that day. Those skilled in the art can understand that for hematopoietic endothelial differentiation and/or endothelial-hematopoietic conversion, which requires a culture process that takes several days, a culture time difference of several hours or more than ten hours will usually not result in the appearance of cell morphology or markers. obvious change.

In the specific embodiments provided herein, the whole process from iPSC to obtaining hematopoietic stem cells is described in detail (Figure 1), especially after inducing hematopoietic mesoderm cells, hematopoietic mesoderm cells or hematopoietic endothelial cells are not separated and purified, but only by changing the composition of the culture medium (and adding DOX) to obtain the final hematopoietic stem cells (including long-term regeneration hematopoietic stem cells). In this process, the effect of adding DOX at different time periods on the generation of hematopoietic endothelial cells and hematopoietic stem cells was studied. It is obvious that those skilled in the art can prepare hematopoietic endothelial cells from mesoderm cells or hematopoietic mesoderm cells based on the technical solutions disclosed herein, or prepare hematopoietic stem cells from mesoderm cells, hematopoietic mesoderm cells or hematopoietic endothelial cells, but as long as the expression of the above-mentioned transcription factors is induced at the corresponding time, these modified technical solutions should also be included in the scope of the present invention.

The inventors established a serum-free differentiation system and an easy-to-operate differentiation process, which stage-specifically regulates key signaling pathways related to the development of hematopoietic stem cells and induces stage-specific interactions with hematopoietic stem cells through the Tet-on tetracycline inducible expression system. The expression of OCT4, a core transcription factor related to development process, achieves efficient differentiation of human pluripotent stem cells into CD34 ⁺ EPCR ⁺ CD90 ⁺ ITGA3 ⁺ long-term regenerative hematopoietic stem cells in vitro. The inventors also found that overexpression of OCT4 promoted the proliferation of CD34 ⁺ EPCR ⁺ CD90 ⁺ ITGA3 ⁺ long-term regenerative hematopoietic stem cells in in vitro amplification culture.

The present invention is described in detail below through specific examples.

Reagents and experimental methods.

Reagents.

Some of the reagent information mentioned in this article is shown in Table 1.

Table 1. Reagent information.

experimental method.

Flow cytometric detection of cell surface markers.

1. Reagents and antibodies required for FACS testing.

(1) Cleaning reagent: Buffer A (PBS+4% FBS).

(2) Directly labeled primary antibodies: FITC anti-human CD34 antibody, APC anti-human KDR antibody, PE anti-human PDGFRα antibody, PE anti-human CD144 antibody, APC anti-human ITGA3 antibody, PE anti-human EPCR antibody, PerCP/Cyanine5.5 anti-human CD90 antibody.

2. Preparation of samples to be tested.

1) Prepare TrypLE working solution: Pipette an appropriate amount of DPBS into a new 15mL centrifuge tube, add the corresponding volume of TrypLE stock solution at a ratio of 1:1, and mix to create the working solution. Preheat the water bath at 37°C for 10 minutes.

2) Take the differentiated cells from the incubator, discard the original culture medium, add an appropriate amount of DPBS to wash the cells, and wash them twice with DPBS (each time The amount of DPBS should not be less than the amount of original culture medium), 1 minute each time (when washing, place DBPS in the plate/bottle for 30 to 45 seconds before aspirating).

3) Add TrypLE working solution (add 1mL TrypLE working solution to each well of the 6-well culture plate) to evenly cover the bottom of the plate, place it in the incubator and incubate for 2 to 5 minutes. During this period, you can observe under the microscope to see that the cells shrink, become round and disperse. Just turn it on.

4) Gently tap the culture flask/plate to detach the cells from the bottom of the plate, then gently blow several times with a pipette, and finally add an equal volume of Buffer A to terminate the digestion. After counting the cells, take 1×10 ⁶ cells. (Suspension cells do not require the cell digestion step, and the suspension cells can be directly collected for subsequent operations).

5) After balancing, centrifuge at 200g for 5 minutes. After centrifugation, discard the supernatant. Flick the bottom of the centrifuge tube to fully disperse the cells. Add an appropriate amount of Buffer A to resuspend. Centrifuge at 200g for 5 minutes and discard the supernatant.

6) Wash the cells twice with Buffer A, 3mL of Buffer A each time, centrifuge at 200g for 5 minutes, and discard the supernatant.

7) Incubate the direct-labeled primary antibody: After resuspending the cells with 100 μL Buffer A, add 1 test direct-labeled primary antibody to each tube, incubate at 4°C for 30 minutes, and flick the centrifuge tube every 10 minutes to fully combine the cells with the antibody.

8) Wash the cells three times with Buffer A, 3 mL of Buffer A each time, centrifuge at 200g for 5 minutes, and discard the supernatant.

9) Add 200 μL DPBS to each tube to resuspend the cells, and filter the cells through a 70 μm pore size filter to remove undigested cell clumps. Transfer to a 96-well culture plate and store in the dark at 4°C until detection on the machine. .

Note:

On the third day after induction of differentiation, hematopoietic mesoderm cell markers KDR and PDGFRα were detected;

Hematopoietic endothelial cell markers CD34, KDR and CD144 were detected on days 6 and 9 of induced differentiation;

Hematopoietic stem progenitor cell markers CD34 and CD45 were detected on days 9 and 12 of induced differentiation;

On the 12th day after induction of differentiation, long-term regenerative hematopoietic stem cell markers CD34, CD90, CD45RA, EPCR and ITGA3 were detected.

Flow cytometric detection of intranuclear markers.

1. Reagents and antibodies required for FACS testing.

(1) Cleaning reagent: Buffer A (PBS+4% FBS).

(2) Punch reagent: Buffer B (PBS+4% FBS+0.4% Triton X-100).

(3) Fixation reagent: PBS+4% paraformaldehyde.

(4) Directly labeled primary antibody: Human/Mouse Brachyury Alexa 488-conjugated Antibody et al.

2. Preparation of samples to be tested.

2) Take the differentiated cells from the incubator, aspirate the original culture medium, add an appropriate amount of DPBS to clean the cells, and wash them twice with DPBS (the amount of DPBS each time is not less than the amount of the original culture medium), 1 minute each time (when washing , place DBPS in the plate/bottle for 30 to 45 seconds before aspirating).

4) Gently tap the culture bottle/plate to make the cells detach from the bottom of the plate, then gently pipet several times with a pipette, and finally add an equal volume of Buffer A to terminate digestion. After counting the cells, take 1×10 ⁶ cells.

5) After balancing, centrifuge at 200g for 5 minutes. After centrifugation, discard the supernatant and flick the bottom of the centrifuge tube to fully disperse the cells. Add 0.5mL PBS+4% paraformaldehyde to each tube and flick the centrifuge tube to suspend the cells in Cells were fixed in paraformaldehyde solution, fixed at 4°C for 15 minutes, centrifuged at 200 g for 5 minutes, and the supernatant was discarded.

6) Wash the cells three times with Buffer B. Buffer B contains 0.4% Triton X-100, which can perforate the cell membrane. Use 3mL of Buffer B each time, and centrifuge at 200g for 5 minutes. Discard the supernatant.

7) Incubate direct-labeled primary antibody: After resuspending the cells with 100 μL Buffer B, add 1 test direct-labeled primary antibody to each tube, incubate at 4°C for 30 minutes, and flick the centrifuge tube every 10 minutes to fully combine the cells with the antibody.

Note: The mesodermal cell marker Brachyury (T) is detected on the first day of induced differentiation.

Streaming computer testing.

1) Turn on the flow cytometer Guava easyCyte HT and the computer.

2) Set up the flow cytometer; open the flow cytometer software and set various parameters.

3) After the machine changes to Ready status, clean the machine.

4) First, set the voltage and gain of FSC and SSC through the isotype control sample so that the discrete cell group is located in the appropriate position of the quadrant. Generally, the lower left corner is the cell debris and the upper right corner is the larger cell clump. Circle the target cell group, set Gate, and proceed to the next step of analysis.

5) Select the appropriate detection channel based on the fluorescein coupled to the antibody. By adjusting the corresponding channel voltage and compensation, the negative cell population and positive cell populations can be clearly distinguished, and then the experimental samples are detected sequentially.

6) After the test is completed, clean the flow cytometer and turn off the flow cytometer and computer.

Example 1 Construction of the stable cell line hiPS-001-5-OCT4.

In order to explore the effect of overexpression of OCT4 on the induction of differentiation, expansion culture and maintenance of stemness of hematopoietic stem cells, we used lentivirus to construct a stable cell line hiPS-001-5-OCT4 (hiPS-001- 5 is the induced pluripotent stem cells prepared by the inventor. For the preparation method, see CN113462638A). The following two expression vectors were used to construct this cell line: TetO-FUW-OCT4-EF1α-NeoR and pLenti-EF1a-rtTA-IRES-PuroR, which each carry a drug screening resistance gene. We used 500 μg/mL G418 and 1 μg/mL Puromycin to conduct drug screening on cells 72 hours after transfection with the virus and obtained the stably transfected cell line hiPS-001-5-OCT4. Then, we characterized the obtained stably transfected cell line hiPS-001-5-OCT4. The experimental results showed that the target gene was integrated into the genomic DNA, and DOX could induce the transcriptional expression of the inserted gene OCT4 (Figure 2, Panel A).

Example 2 OCT4 expression promotes the generation of long-term regenerative hematopoietic stem cells.

OCT4 is a key transcriptional cytokine of pluripotent stem cells, combining with SOX2, NANOG, etc. to maintain cell pluripotency and cell self-renewal (Babaie et al., 2007; Greco et al., 2007; Zhou et al., 2007). OCT4 combined with SOX2, NANOG, c-MYC or Lin28 can reprogram fibroblasts into pluripotent stem cells (Takahashi et al., 2007; Yu et al., 2007). Studies have shown that the histone deacetylation inhibitor Valproic acid (VPA) promotes the in vitro expansion of cord blood CD34 ⁺ and CD34 ⁺ CD90 ⁺ cells, and causes the cells to upregulate the expression of pluripotent stem cell genes OCT4, Nanog, SOX2, ZIC3, etc. Using siRNA to knock down OCT4, Nanog, SOX2, and ZIC3 genes inhibited the in vitro expansion effect of VPA on cord blood CD34 ⁺ and CD34 ⁺ CD90 ⁺ cells (Chaurasia et al., 2014). The small molecule compound OCT4-activating compound 1 (OAC1) can activate the expression of endogenous OCT4 in cells and promote the expansion of cord blood-derived CD34 ⁺ hematopoietic stem and progenitor cells (Huang et al., 2016). Another study reported that combined with related cytokine treatment, overexpression of OCT4 can reprogram fibroblasts into CD45 ⁺ hematopoietic cells, indicating that OCT4 plays an important role in hematopoietic cell fate determination (Szabo et al., 2010). However, long-term or high-level overexpression of OCT4 inhibits the hematopoietic differentiation of ESCs (Camara-Clayette et al., 2006).

In order to study the effect of stage-specific expression of OCT4 on hematopoietic stem cell differentiation, we added DOX to induce the expression of OCT4 gene at different time windows during induction of differentiation. Analysis of the flow cytometry results on the 6th day of differentiation showed that the induction efficiency of CD34 ⁺ KDR ⁺ hematopoietic endothelial cells in the Control control group was 47.46%; while in the experimental group that added DOX on the 3rd to 6th day of differentiation, the induction efficiency of CD34 ⁺ KDR ⁺ hematopoietic endothelial cells was 47.46%. The induction efficiency was reduced to 11.98%. The above experimental results show that early overexpression of OCT4 is not conducive to the induction of differentiation of hematopoietic endothelial cells (Figure 2, Panel B). To examine the effect of overexpression of OCT4 on induced differentiation of hematopoietic cells, we examined the expression of CD34 ⁺ CD45 ⁺ hematopoietic cells. Flow cytometry results on the 9th day of differentiation showed that adding DOX to D3-9 and D6-9 resulted in a decrease in CD34 ⁺ cell differentiation efficiency, but significantly increased the generation of CD45 ⁺ cells, and D6-9 treatment of DOX significantly increased CD34 ⁺ CD45 ⁺ hematopoiesis. Induction efficiency of cells (panel C of Figure 2). The above experimental results show that overexpression of OCT4 promotes the generation of CD45 ⁺ hematopoietic cells. We further analyzed the effect of overexpression of OCT4 on the generation of long-term regenerative hematopoietic stem cells. The flow cytometry results on the 12th day of differentiation showed that D6-9, D6-12 and D9-12 treated DOX to improve the differentiation efficiency of CD34 ⁺ EPCR ⁺ CD90 ⁺ ITGA3 ⁺ long-term regenerative hematopoietic stem cells, and the better time window for DOX treatment is D9-12 (D-G in Figure 2).

The above experimental results indicate that premature activation of OCT4 inhibits the differentiation of CD34 ⁺ KDR ⁺ hematopoietic endothelial cells, but activation of OCT4 in the late differentiation stage promotes the generation of CD34 ⁺ CD45 ⁺ hematopoietic cells and promotes hematopoietic cells to express the key marker genes CD34, EPCR, CD90 and ITGA3 for long-term regeneration of hematopoietic stem cells.

Example 3 OCT4 expression promotes the maintenance of long-term regenerative hematopoietic stem cells.

Relevant studies have shown that activation of OCT4 by the small molecule compound OAC1 can promote the expansion and culture of CD34 ⁺ hematopoietic stem and progenitor cells in vitro (Huang et al., 2016). Knockdown of OCT4 inhibits the in vitro expansion effect of VPA on cord blood CD34 ⁺ and CD34 ⁺ CD90 ⁺ cells (Chaurasia et al., 2014). The above studies suggest that our activation of OCT4 may be an important factor in the maintenance and culture of hematopoietic stem and progenitor cells in vitro. In order to achieve the expansion and stemness maintenance of hematopoietic stem cells in vitro, we tested the effect of DOX-induced OCT4 gene overexpression on hematopoietic stem cell culture. The starting cells on Day0 here are hematopoietic stem and progenitor cells. The cells obtained on Day12 based on the differentiation process in Figure 1 are hematopoietic stem and progenitor cells (but D9-12 does not need to induce OCT4 expression), and then the cells obtained at this time are The beginning of a new experiment. The experimental results showed that after 3 days of in vitro expansion and culture, the number of cells increased significantly. Although DOX treatment caused a reduction in the fold of cell proliferation compared with the control group, it improved CD34 ⁺ EPCR ⁺ CD90 ⁺ ITGA3 ⁺ long-term regeneration. The proportion of hematopoietic stem cells. Further analysis showed that DOX treatment increased the absolute cell number of CD34 ⁺ EPCR ⁺ CD90 ⁺ ITGA3 ⁺ long-term regenerating hematopoietic stem cells, while the absolute cell number of CD34 ⁺ EPCR ⁺ CD90 ⁺ ITGA3 ⁺ long-term regenerating hematopoietic stem cells in the control group was significantly reduced (Figure Picture A-B of 3).

The above experimental results show that in short-term in vitro expansion culture, overexpression of OCT4 promotes the proliferation of CD34 ⁺ EPCR ⁺ CD90 ⁺ ITGA3 ⁺ long-term regenerative hematopoietic stem cells.

Example 4 Human pluripotent stem cells induce the differentiation process of hematopoietic stem cells.

4.1 Formation of a single layer of adherent cells.

Experimental operation: Day-1.

1) Take an appropriate amount of TrypLE working solution and place it in a 37°C water bath to preheat for 10 minutes.

2) According to the amount of culture medium required for passage, prepare TeSR-E8 culture medium containing 10 μM Y-27632, and add 1 μL Y-27632 (10mM) storage solution to every ml of TeSR-E8 culture medium. Preheat the water bath at 37°C for 10 minutes.

3) Take out the hiPSC-001-5-OCT4 cells to be passaged from the incubator (cell confluence is 70% to 80%) (see Figure 4 for cell morphology), discard the original culture medium, and wash it twice with DPBS (DPBS each time) The dosage should not be less than the amount of original culture medium), 1 minute each time (when washing, place DBPS in the well/bottle for 30 to 45 seconds before aspirating).

4) After adding TrypLE working solution (approximately 1mL TrypLE working solution for six-well plates, and approximately 2mL TrypLE working solution for T25 bottles), make it evenly cover the bottom of the plate, place it in the incubator and incubate for 2 to 5 minutes. During this period, you can observe under the microscope. , the cells can shrink, become round and spread out.

5) Gently tap the culture bottle/plate to make the cells detach from the bottom of the plate, then gently pipet several times with a pipette, and finally add an equal volume of digestion stop solution to terminate digestion.

6) After balancing, centrifuge at 200g for 5 minutes. After centrifugation, discard the supernatant and flick the bottom of the centrifuge tube to fully disperse the cells. Add an appropriate amount of TeSR-E8 culture medium containing 10 μM Y-27632 to resuspend. After counting the cells, adjust to Suitable cell density.

7) Take out the Matrigel-coated culture plate/bottle, remove the remaining coating solution, and wash it once with DPBS. Inoculate the evenly mixed cell suspension into the coated culture plate/bottle at a density of 8000 cells/ ^cm2 , and mark the passage date, cell type, cell generation and other information. Place the culture plate/bottle in a 37°C, 5% _CO2 incubator for static culture. Record as Day-1.

Note: The cell seeding density should be controlled at 8000-10000 cells/cm ² . Do not shake the culture plate/bottle after seeding to prevent cells from gathering in the center of the culture plate/dish.

4.2 Mesoderm Induction.

Experimental operation: Day0.

1) Take an appropriate amount of mesoderm induction medium and place it in a 37°C water bath to preheat for 10 minutes.

2) After 24 hours of formation of a monolayer of adherent cells, take out the cells to be differentiated from the incubator (see Figure 5 for cell morphology), discard the original culture medium, add an appropriate amount of DPBS to wash the cells, and wash twice with DPBS (DPBS each time) The dosage should not be less than the amount of the original culture medium), 1 minute each time (when washing, place DBPS in the plate/bottle for 30 to 45 seconds before aspirating).

3) Add mesoderm induction medium, and then place in a 37°C, 5% CO ₂ incubator for 24 hours (add 2 mL of culture medium to each well of a 6-well culture plate).

4.3 Hematopoietic mesoderm specification.

Experimental operation: Day 1.

1) Prepare an appropriate amount of specialized culture medium for hematopoietic mesoderm and place it in a 37°C water bath to preheat for 10 minutes.

2) After 24 hours of mesoderm induction, take the differentiated cells from the incubator (see Figure 6 for cell morphology, and see Figure 7 for flow cytometric detection results of mesoderm markers). Aspirate the original culture medium, add an appropriate amount of DPBS to wash the cells, and wash the cells with DPBS. Wash twice (the amount of DPBS each time is not less than the amount of original culture medium), 1 minute each time (when washing, place DBPS in the plate/bottle for 30 to 45 seconds before aspirating).

3) Add hematopoietic mesoderm specialized medium, and then place it in a 37°C, 5% CO ₂ incubator for 48 hours (add 2 mL of culture medium to each well of a 6-well culture plate).

4.4 Hematopoietic endothelium specification (hemogenic endothelium specification) and endothelial-to-hematopoietic transition (ETH): Day3.

Experimental operation: Day3.

1) Prepare an appropriate amount of hematopoietic endothelial specialization and endothelial-hematopoietic cell transformation medium, and place it in a 37°C water bath to preheat for 10 minutes.

2) Take an appropriate amount of TrypLE working solution and place it in a 37°C water bath to preheat for 10 minutes.

3) After 48 hours of hematopoietic mesoderm specialization, take the differentiated cells from the incubator (see Figure 8 for cell morphology, and see Figure 9 for the flow cytometric detection results of hematopoietic mesoderm markers). Aspirate the original culture medium, and add an appropriate amount of DPBS to clean the cells. And wash twice with DPBS (the amount of DPBS each time is not less than the amount of original culture medium), 1 minute each time (when washing, place DBPS in the plate/bottle for 30 to 45 seconds before aspirating).

4) Add TrypLE working solution (add 1mL TrypLE working solution to each well of the 6-well culture plate) to evenly cover the bottom of the plate, place it in the incubator and incubate for 2 to 5 minutes. During this period, you can observe under the microscope to see that the cells shrink, become round and disperse. Just turn it on.

5) Gently tap the culture bottle/plate to make the cells detach from the bottom of the plate, then gently pipet several times with a pipette, and finally add an equal volume of termination digestion solution to terminate digestion.

6) After balancing, centrifuge at 200g for 5 minutes. After centrifugation, discard the supernatant, tap the bottom of the centrifuge tube to fully disperse the cells, add an appropriate amount of hemogenic endothelial specialization and endothelial-hematopoietic cell conversion medium containing 10μM Y-27632 to resuspend, count the cells, and adjust to an appropriate cell density.

7) Take out the matrigel-coated culture plate/bottle, remove the remaining coating solution, and wash it once with DPBS. Inoculate the evenly mixed cell suspension into the coated culture plate/bottle at a seeding density of 2×10 ⁴ cells/cm ² . Mark the passage date, cell type, cell generation and other information, and then place it at 37°C and 5% Cultivate statically in a _CO2 incubator (add 2 mL of culture medium to each well of a 6-well culture plate).

Experimental operation: Day4.

2) Take the differentiated cells from the incubator (see Figure 10 for cell morphology), discard the original culture medium, replace it with fresh hematopoietic endothelial specialization and endothelial-hematopoietic cell transformation medium, and then culture it at 37°C and 5% _CO2 Cultivate statically in the box (add 2 mL of culture medium to each well of a 6-well culture plate).

Experimental operation: Day6.

1) Prepare an appropriate amount of hematopoietic endothelial specialization and endothelial-hematopoietic cell transformation medium containing 5 μg/mL Doxycycline, and place it in a 37°C water bath to preheat for 10 minutes.

2) Take the differentiated cells from the incubator (see Figure 11 for cell morphology, and see Figure 12 for the flow cytometric detection results of hematopoietic endothelial cell markers), aspirate the original culture medium, and replace it with fresh hematopoietic endothelial differentiation and endothelial cells containing 5 μg/mL Doxycycline. - Hematopoietic cell transformation medium, and then placed in a 37°C, 5% CO ₂ incubator for static culture (add 2 mL of culture medium to each well of a 6-well culture plate).

Experimental operation: Day8.

2) Take the differentiated cells from the incubator, aspirate the original culture medium, replace with fresh hematopoietic endothelial specialization and endothelial-hematopoietic cell transformation medium, and then place it in a 37°C, 5% CO ₂ incubator for static culture (6 Add 2 mL of culture medium to each well of the well culture plate). Mark it as Day8.

3) On Day 9, the differentiated cells were taken out of the incubator, the original culture medium was discarded, and the fresh hemogenic endothelial specialization and endothelial-hematopoietic cell conversion medium containing 5 μg/mL Doxycycline was replaced, and then placed in a 37°C, 5% _CO2 incubator for static culture (2 mL of culture medium was added to each well of a 6-well culture plate).

4) On Day 9, observe the cell morphology and detect the expression of hematopoietic endothelial cell markers CD34, KDR and CD144 by flow cytometry. For specific detection methods, please refer to the cell flow cytometry in the Experimental Methods section. The results are shown in Figure 13 and Figure 14 respectively.

Note: In the hematopoietic endothelial stage, hematopoietic endothelial cells migrate to form a hematopoietic center, and a small number of suspended cells appear. Flow cytometry detects hematopoietic endothelial cell markers CD34, KDR and CD144. The proportion of CD34 ⁺ KDR ⁺ cells should be no less than 30%, and the proportion of CD144 ⁺ cells among CD34 ⁺ KDR ⁺ cells should be no less than 80%.

Experimental operation: Day10.

2) Take the differentiated cells from the incubator, collect the original culture medium into a 15mL centrifuge tube, balance and centrifuge at 200g for 5 minutes. After centrifugation, discard the supernatant, tap the bottom of the centrifuge tube to fully disperse the cells, add an appropriate amount of hemogenic endothelial specialization and endothelial-hematopoietic cell conversion medium containing 5μg/mL Doxycycline and resuspend them.

3) Re-inoculate the resuspended cells into the culture plate/bottle, and then place them in a 37°C, 5% CO ₂ incubator for static culture (add 2 mL of culture medium to each well of the 6-well culture plate).

Experimental operation: Day12.

Take the differentiated cells from the incubator (the flow cytometric detection results of cell morphology and long-term regeneration hematopoietic stem cell markers CD34, CD90, EPCR and ITGA3 are shown in Figure 15 and Figure 16 respectively), collect the original culture medium into a 15mL centrifuge tube , balance and centrifuge, centrifuge at 200g for 5 minutes, discard the supernatant after centrifugation, flick the bottom of the centrifuge tube to fully disperse the cells, and use the obtained hematopoietic stem cells for subsequent experiments or cryopreservation.

Example 5 Verification of differentiation potential of hematopoietic stem cells.

Colony-Forming Unit Assays (CFU, Colony-Forming Unit Assays) verify the in vitro differentiation potential of hematopoietic stem cells.

The hematopoietic stem cells induced and differentiated by hiPS001-5-OCT4 in Example 4 can successfully form multi-lineage progenitor cells (CFU-GEMM) and granulocytes (CFU-G) after being cultured in methylcellulose medium for 14 days. , macrophage (CFU-M), granulocyte/macrophage (CFU-GM), red blood cell (B/C-FUE) colony unit (Figure 17).

Claims

The method for preparing hematopoietic stem cells or hematopoietic stem progenitor cells is characterized by including:

1) Provide hematopoietic mesoderm cells or cell culture including hematopoietic mesoderm cells;

2) Culturing the hematopoietic mesoderm cells or cell culture including hematopoietic mesoderm cells in a hematopoietic endothelial specialization and endothelial-hematopoietic conversion medium; and

3) Let the hematopoietic mesoderm cells express or overexpress the transcription factor OCT4.
The method of claim 1, wherein step 3) is performed 3 days after step 2).
The method of claim 1 or 2, wherein step 3) is performed for at least 4 days.
The method according to claim 1 or 2, wherein step 3) is performed on the 4th to 7th day, the 4th to 10th day or the 7th to 10th day of the culture in step 2).
The method of claim 1 or 2, wherein the hematopoietic endothelial specialization and endothelial-hematopoietic conversion medium contains VEGF, bFGF, SCF, IL-3, TPO, Flt-3L and BMP4.
The method according to claim 5, characterized in that the hemogenic endothelial specialization and endothelial-hematopoietic transition medium is a STEMdiffTMAPELTM2 medium supplemented with VEGF, bFGF, SCF, IL-3, TPO, Flt-3L and BMP4.
The method according to claim 1 or 2, characterized in that the hematopoietic mesoderm cells include an exogenously introduced nucleic acid sequence encoding the transcription factor OCT4;

The coding nucleic acid sequence is operably linked to an inducible promoter;

The inducible promoter is a tetracycline-inducible promoter.
The method of claim 7, wherein the hematopoietic mesoderm cells further comprise an exogenously introduced rtTA encoding nucleic acid sequence; the encoding nucleic acid sequence is integrated into the genome of the hematopoietic mesoderm cells.
The method of claim 1 or 2, wherein step 3) allows the hematopoietic mesoderm cells to express by adding tetracycline or doxycycline to the hematopoietic endothelial specialization and endothelial-hematopoietic conversion medium. or overexpression of the transcription factor OCT4.
The method of claim 1 or 2, wherein the hematopoietic mesoderm cells or the cell culture comprising hematopoietic mesoderm cells are produced by allowing the mesoderm cells or the cell culture comprising mesoderm cells to grow in the hematopoietic mesoderm. Obtained by culturing in specialized media.
The method of claim 10, wherein the hematopoietic mesoderm is obtained by culturing the mesoderm cells or the cell culture including mesoderm cells in the hematopoietic mesoderm specialized medium for 2 days. cells or cell cultures including hematopoietic mesodermal cells.
The method of claim 10, wherein the hematopoietic mesoderm specialized medium contains VEGF and bFGF.
The method of claim 12, wherein the hematopoietic mesoderm specialized medium is STEMdiffTMAPELTM2 medium supplemented with VEGF and bFGF.
The method according to claim 1 or 2, characterized in that:

The hematopoietic mesoderm cells are KDR + and PDGFRα- ;

The hematopoietic stem cells are CD34 + CD45RA - CD90 + EPCR + ;

The hematopoietic stem and progenitor cells are CD34 + and CD45 + .
The method of claim 1 or 2, wherein the hematopoietic stem cells are long-term regenerative hematopoietic stem cells, and the long-term regenerative hematopoietic stem cells are CD34 + EPCR + CD90 + ITGA3 + .
A method for culturing the long-term regenerative hematopoietic stem cells of claim 15 in a culture medium or culturing a cell culture including the long-term regenerative hematopoietic stem cells of claim 15, characterized in that it includes allowing the long-term regenerative hematopoietic stem cells to express or overexpress Cytokine OCT4.
The method of claim 16, wherein the expression or overexpression of the cytokine OCT4 is performed on days 1-3 of culturing the long-term regenerative hematopoietic stem cells or a cell culture comprising long-term regenerative hematopoietic stem cells.
The method of claim 16 or 17, wherein the expression or overexpression of the cytokine OCT4 is performed in one of the following ways:

1) Adding a cytokine OCT4 expression activator to the culture medium; and

2) Introducing the exogenous nucleic acid sequence encoding the transcription factor OCT4 into the long-term regenerative hematopoietic stem cells.
The method of claim 18, wherein the cytokine OCT4 expression activator is OAC1.
The method of claim 18, wherein the coding nucleic acid sequence is operably linked to an inducible promoter.
The method of claim 20, wherein the inducible promoter is a tetracycline-inducible promoter.
The method of claim 18, wherein the long-term regenerative hematopoietic stem cells further comprise an exogenously introduced rtTA encoding nucleic acid sequence.