Method for proliferating and / or differentiating T cells

Abstract

The present application relates to a method for proliferating and / or differentiating T cells, comprising the steps of culturing pluripotent cells using an Rho activator I / II, and proliferating and / or differentiating the pluripotent cells into T cells. The invention further provides a culture medium for culturing the pluripotent cells and proliferating and / or differentiating the pluripotent cells into the T cells, a culture platform and application.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biological medicine, in particular to a method for proliferating and / or differentiating T cells, a culture medium used in the method and application thereof. BACKGROUND

[0002] Human induced pluripotent stem cells (iPSCs) have unlimited proliferation capacity and can differentiate into all types of cells in the body, including hematopoietic stem cells for cell therapy and T, B, NKT and NK lymphocytes, and are an ideal source of allogeneic immune cells. iPSCs can also be genetically modified to have enhanced specificity and effector function, producing large amounts of tumor antigen-specific T and NK cells in vitro, and enhancing the persistence after reinfusion in vivo. At present, the process of inducing iPSCs to produce immune cells in vitro usually uses stepwise addition of small molecules or cell growth factors to activate important signaling pathways, to simulate a series of blood and immune system development processes during embryonic and fetal periods. Among them, whether hematopoietic stem cells with lymphocyte differentiation potential can be induced is a very important intermediate link. This is because there are at least two waves of temporary and spatially unique blood formation during embryonic development: primitive hematopoiesis and definitive hematopoiesis. Among them, only during the development of definitive hematopoiesis, the newly formed hematopoietic stem cells from arterial endothelial cells, after a series of development and maturation processes, can maintain adult multilineage hematopoiesis and generate T, B, NK and NKT lymphocytes, etc. Although various reported differentiation protocols can generate hematopoietic stem cells from human iPSCs, most of the protocols promote primitive hematopoiesis, and the generated hematopoietic stem cells cannot long-term self-renew, multi-lineage differentiation, do not have engraftment potential, and cannot be effectively differentiated into T cells and NK cells and other lymphocytes.

[0003] Therefore, the reported protocols for inducing iPSCs to generate T and NK cells and other lymphocytes have low yield, complex production process (sorting is required during induction, which increases experimental difficulty), low safety (interference of foreign substances, feeder cells), and other difficulties, which seriously hinder the industrialization and large-scale production and application.

[0004] Current protocols for iPSCs in vitro differentiation into T cells and NK cells mostly use 3D suspension culture method of embryoid bodies (EB) at hematopoietic stem cell stage, which requires cells to be settled and aggregated in microplate by centrifugation or iPSCs to be passively aggregated and spontaneously form spheroids in suspension culture. But the former method is complicated, time-consuming and labor-intensive, and is not suitable for large-scale expansion and use, and the latter method has uneven spheroid size, increased cell heterogeneity, and unstable yield and purity. In addition, most protocols need to dissociate spheroids into single cells at the end of differentiation to obtain CD34+ HPCs, which can enter subsequent differentiation steps, usually with very low yield and complicated operation.

[0005] So far, the molecular mechanism of hematopoietic progenitor / stem cells developing into T cells in vivo has not been fully elucidated, and the traditional in vitro differentiation method of T cells uses trophoblast cells or animal-derived components to maintain and induce cell differentiation, and the composition of the culture medium is unclear, which far cannot meet the safety requirements of biological products. Now there are also a few differentiation methods that can differentiate T cells in serum-free and trophoblast-free systems, but these methods cannot efficiently obtain late and mature T cells.

[0006] Studies have shown that the production of hematopoietic stem cells and T, NK and other lymphocytes from pluripotent stem cells depends on how to promote early embryonic hematopoietic development to differentiate into permanent hematopoietic direction through fully designed and verified methods. In order to meet the industrial demand for efficient production of late and mature T cells, it is urgent to develop a method for inducing iPSCs to differentiate into T cells. SUMMARY

[0007] The present application provides a method for proliferating and / or differentiating T cells, which comprises culturing pluripotent cells with Rho activator I / II, and proliferating and / or differentiating the pluripotent cells into T cells.

[0008] The method provided by the present application has one or more of the following advantages:

[0009] (1) The hematopoietic stem cell differentiation protocol used involves culturing cells on a shaker throughout the entire process. On the second day after iPSC single-cell inoculation, uniform EB spheres can spontaneously form. Starting from the sixth day of differentiation, HPCs are gradually released into the suspension system. At the end of differentiation, only the suspension cells can be collected without digesting the EB spheres to obtain high-purity CD34+ HPCs. The expansion factor is 5 to 10 times, and they can be directly used for subsequent T and NK differentiation experiments without sorting. (2) When the differentiation system is expanded, the differentiation efficiency is high and reproducible. It can be easily applied to commercially available conical flasks or other large-scale cell production devices. Cell sampling and harvesting are easy at any time during the process. (3) By adding the small molecule compound CN03, the production of hematopoietic stem cells with lymphocyte differentiation potential can be effectively promoted. At the same time, it can also significantly promote the generation of terminal T and NK cells and increase their yield. In particular, each iPSC can generate a large number of late mature T cells through differentiation, which have the typical T cell characteristics of CD5+CD8αβ+ and have a high expansion capacity in vitro.

[0010] On one hand, this application provides a method for proliferating and / or differentiating T cells, comprising culturing pluripotent cells using Rho activator I / II and proliferating and / or differentiating the pluripotent cells into T cells, wherein the pluripotent cells may be induced pluripotent stem cells (iPSCs), hematopoietic stem / progenitor cells (HPCs), or modified cells.

[0011] In some embodiments, the Rho activator I / II may be selected from one or more activators of the group consisting of CN02, CN03, and CN04. In some embodiments, the Rho activator II is CN02. In some embodiments, the Rho activator II is CN03. In some embodiments, the Rho activator I is CN04.

[0012] In some embodiments, the method includes culturing pluripotent cells using Rho activator I / II for 12-96 hours. In some embodiments, the method includes culturing pluripotent cells using Rho activator I / II for 12-72 hours. In some embodiments, the method includes culturing pluripotent cells using Rho activator I / II for 12-48 hours. In some embodiments, the method includes culturing pluripotent cells using Rho activator I / II for 24-48 hours. In some embodiments, the method includes culturing pluripotent cells using CN03 for approximately 48 hours.

[0013] In some embodiments, the concentration of Rho activator I / II is 50-750 ng / mL. In some embodiments, the concentration of Rho activator I / II is 150-350 ng / mL. In some embodiments, the concentration of Rho activator I / II is about 250 ng / mL.

[0014] In some embodiments, the concentration of CNO3 is 50-750 ng / mL. In some embodiments, the concentration of CNO3 is 150-350 ng / mL. In some embodiments, the concentration of CNO3 is about 250 ng / mL.

[0015] In some embodiments, the T cells are CD5+CD7+ T precursor cells. In some embodiments, the T cells are CD5+CD8αβ+ T cells.

[0016] In some embodiments, the method includes culturing cells under hypoxic conditions. In some embodiments, the hypoxic conditions are 5% O2. In some embodiments, the method includes culturing cells under serum-free culture conditions. In some embodiments, the method does not use feeder cells.

[0017] In some embodiments, the method includes culturing pluripotent cells using a culture medium containing one or more of the following factors: CHIR99021, Y-27632, GlutaMAX, L-ascorbic acid, MTG, ITS, BMP4, VEGF, bFGF, SB 431542, SCF, TPO, FLT3, Sodium Pyruvate, β-Mercaptoethanol, NEAA, IL-7, IL-15, SB203580, SDF-1α, Dexamethasone, IL-21, and Anti-CD3 OKT3.

[0018] In some embodiments, the method includes culturing pluripotent cells using a culture medium comprising a basal medium. In some embodiments, the basal medium is selected from one or more of the following: Nuwacell® ncTarget complete medium, Stempro-34 complete medium, and StemSpan SFEM II medium.

[0019] On the other hand, this application provides a culture medium for culturing pluripotent cells and differentiating them into T cells, the culture medium containing Rho activator I / II.

[0020] In some embodiments, the pluripotent cells in the culture medium are induced pluripotent stem cells (iPSCs). In some embodiments, the pluripotent cells in the culture medium are hematopoietic stem / progenitor cells (HPCs). In some embodiments, the pluripotent cells in the culture medium are modified cells.

[0021] In some embodiments, the Rho activator I / II in the culture medium is selected from one or more activators of the group consisting of CNO2, CNO3, and CNO4. In some embodiments, the Rho activator II in the culture medium is CNO2. In some embodiments, the Rho activator II in the culture medium is CNO3. In some embodiments, the Rho activator I in the culture medium is CNO4.

[0022] In some embodiments, the concentration of Rho activator I / II in the culture medium is 50-750 ng / mL. In some embodiments, the concentration of Rho activator I / II in the culture medium is 150-350 ng / mL. In some embodiments, the concentration of Rho activator I / II in the culture medium is about 250 ng / mL. In some embodiments, the concentration of CNO3 in the culture medium is 50-750 ng / mL. In some embodiments, the concentration of CNO3 in the culture medium is 150-350 ng / mL. In some embodiments, the concentration of CNO3 in the culture medium is about 250 ng / mL.

[0023] In some embodiments, the T cells in the culture medium are CD5+CD7+ T precursor cells. In some embodiments, the T cells in the culture medium are CD5+CD8αβ+ T cells.

[0024] In some embodiments, the culture medium contains one or more of the following factors: CHIR99021, Y-27632, GlutaMAX, L-ascorbic acid, MTG, ITS, BMP4, VEGF, bFGF, SB 431542, SCF, TPO, FLT3, Sodium Pyruvate, β-Mercaptoethanol, NEAA, IL-7, IL-15, SB203580, SDF-1α, Dexamethasone, IL-21, and Anti-CD3 OKT3.

[0025] In some embodiments, the culture medium includes a basal culture medium selected from one or more of the following: Nuwacell® ncTarget complete medium, Stempro-34 complete medium, and StemSpan SFEM II medium.

[0026] On the other hand, this application provides a composition comprising pluripotent cells and the culture medium described in this application.

[0027] On the other hand, this application provides a culture platform for obtaining T cells, which includes the methods described in this application and / or the culture medium described in this application.

[0028] Other aspects and advantages of this application will readily be apparent to those skilled in the art from the detailed description below. Only exemplary embodiments of this application are shown and described in the following detailed description. As will be appreciated by those skilled in the art, the content of this application enables them to make modifications to the disclosed specific embodiments without departing from the spirit and scope of the invention to which this application pertains. Accordingly, the descriptions in the accompanying drawings and specification of this application are merely exemplary and not restrictive. Attached Figure Description

[0029] The features and advantages of the invention related to this application can be better understood by referring to the exemplary embodiments and accompanying drawings described in detail below. A brief description of the drawings is as follows:

[0030] Figure 1 shows the flowchart (Figure A), morphology (Figure B), cell phenotype and yield (Figure CF) of the iPSC-HPCs 3D differentiation method described in Example 1 of this application.

[0031] Figure 2 shows the effects of the small molecule additives CN03 and Endothelin-1 (EDN) described in Example 2 of this application on the phenotype and yield of iPSC-differentiated HPCs. Figures 2A-2B The figure shows the effects of adding 250 ng / mL CN03 and 10 ng / mL and 100 ng / mL Edothelin-1 at different time points on the phenotype and yield of iPSCs to iHPCs, as described in this application. Figures 2C-2D The figure shows the effect of adding different concentrations of CN03 on the phenotype and yield of iPSCs to iHPCs during D5-D7 as described in this application.

[0032] Figure 3 This illustrates the experimental protocol for further differentiating iHPCs into iT cells as described in Example 3 of this application.

[0033] Figure 4 shows the effects of the small molecule additives CN03 and Endothelin-1 (EDN) described in Example 3 of this application on the phenotype (4A), purity (4B), and yield (4C) of iHPCs differentiating into T cell precursors.

[0034] Figure 5 shows the effects of the small molecule additives CN03 and Endothelin-1 (EDN) described in Example 3 of this application on the phenotype (5A) and yield (5B) of mature iT cells generated by iHPCs.

[0035] Figure 6 The display shows the expansion fold at each stage when iPSCs generate mature iT cells after adding small molecule CN03 as described in Example 3 of this application.

[0036] Figure 7 shows the scheme (7A) and phenotype (7B) for generating NK cells using iHPCs as described in Embodiment 4 of this application. Detailed Implementation

[0037] The following specific embodiments illustrate the implementation of the invention. Those skilled in the art can easily understand other advantages and effects of the invention from the content disclosed in this specification.

[0038] Terminology Definition

[0039] In this application, the term "pluripotent cell" generally refers to a cell with the potential for proliferation and differentiation. A pluripotent cell can be functionally defined as a cell that: (1) has the ability to differentiate into different cell types, and in some cases, generates only one specialized cell type; and (2) is capable of long-term self-renewal, producing one or more cells that are the same as or different from the original cell type. The source and preparation method of pluripotent cells are not limited. For example, the pluripotent cells can be naturally obtained or artificially modified. For example, the pluripotent cells can include induced pluripotent stem cells, hematopoietic stem / progenitor cells, etc.

[0040] In this application, the term "induced pluripotent stem cells" may be abbreviated as "iPS," "iPSC," or "iPSCs," and generally refers to a type of pluripotent stem cells artificially prepared from non-pluripotent cells. Induced pluripotent stem cells can be obtained by introducing specific transcription factors to reprogram terminally differentiated somatic cells. For example, the terminally differentiated cells may be T cells, NK cells, etc.

[0041] In this application, the term "hematopoietic stem / progenitor cells" may be abbreviated as "HPC" or "HPCs," and generally refers to cells with long-term self-renewal capacity and the potential to differentiate into various types of mature blood cells. The source and preparation method of hematopoietic stem / progenitor cells are not limited; for example, the hematopoietic stem cells may be differentiated from pluripotent cells and may be isolated from bone marrow or blood. Hematopoietic stem cells can differentiate into various cell types, such as bone marrow lineage cells (e.g., monocytes and macrophages, neutrophils, basophils, eosinophils, erythrocytes, megakaryocytes / platelets, dendritic cells), and lymphoid lineage cells (e.g., T cells, B cells, NK cells).

[0042] In this application, the terms "Rho activator I / II" and "Rho Activator I / II" are used interchangeably and are generally used to refer to the activation of Rho GTPases. The main component of Rho activator II may be the CN03 protein. In this application, Rho activator I / II may be CN02, CN03, or CN04.

[0043] In this application, the term "T cell" generally refers to a cell with the same or similar phenotype as thymus-derived T cells, which can participate in various cell-mediated immune responses. In this application, the term encompasses both unmodified T cells and modified T cells. For example, the T cells may be thymocytes, naive T lymphocytes, immature T lymphocytes, mature T lymphocytes, resting T lymphocytes, activated T lymphocytes, and helper T cells (HTL; CD4+). + T cells, cytotoxic T cells (CTL; ​​CD8) + T cells, CD4 + CD8 + T cells, CD4 - CD8 - T cells, and any other subsets of T cells. For example, the T cells may be CAR-T cells and TCR-T cells. In this application, the T cells may be CD5+CD7+ T cell precursors or CD5+CD8αβ+ T cells.

[0044] In this application, the term "modified" generally refers to an alteration in the state or structure of the cells described in this application. Such modification can be artificial or can be caused by environmental conditions resulting in a change compared to the natural source. In this application, the modification can be carried out in various ways known to those skilled in the art, for example, by physical, biological, chemical, or other methods, modifying the cells structurally and / or functionally. For example, cells can be modified by introducing nucleic acids. For example, cells can be modified by genetic engineering. For example, in this application, the T cells can comprise modified T cells; for example, modified T cells can be TCR-T cells or CAR-T cells. For example, the modified cells can be pluripotent cells, induced pluripotent stem cells, or hematopoietic stem / progenitor cells.

[0045] In this application, the term "composition" generally refers to a product comprising a specified amount of a specified ingredient, and any product produced directly or indirectly from a combination of the specified amounts of the specified ingredients. In this application, the composition may also contain other inactive ingredients, such as carriers, excipients, adjuvants, stabilizers, etc.

[0046] In this application, the term "ex vivo" generally refers to manipulation of cells, tissues, and / or organs that have been removed from a living organism. In some embodiments, the cells, tissues, and / or organs may be returned to the living organism or introduced into another organism by certain methods.

[0047] In this application, the term "in vitro" generally refers to the removal or release of a part of an organism from the organism.

[0048] In this application, the term "comprising" generally means including, encompassing, containing, or including. In some cases, it also means "to be" or "composed of".

[0049] In this application, the term "and / or" should be understood to mean any one, two, or more of the alternatives or any combination thereof.

[0050] In this application, the term "about" generally refers to a variation within a range of 0.5% to 10% above or below a specified value, such as a variation within a range of 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, or 10% above or below a specified value. Invention Details

[0052] Methods

[0053] On one hand, this application provides a method for proliferating and / or differentiating T cells, which includes the steps of culturing pluripotent cells using Rho activator I / II and proliferating and / or differentiating the pluripotent cells into T cells.

[0054] In this application, the method may further include culturing, proliferating, and / or differentiating pluripotent cells; the method may further include culturing and / or differentiating embryomorphic bodies (EBs); the method may further include culturing, proliferating, and / or differentiating hematopoietic stem / progenitor cells; the method may further include culturing, proliferating, and / or differentiating induced pluripotent stem cells; the method may further include culturing T cells; the method may further include proliferating and / or differentiating pluripotent cells into embryomorphic bodies (EBs); the method may further include proliferating and / or differentiating pluripotent cells into hematopoietic stem / progenitor cells; the method may further include differentiating embryomorphic bodies (EBs) into T cells; the method may further include proliferating and / or differentiating hematopoietic stem / progenitor cells into T cells.

[0055] In this application, the culture method may involve culturing cells on a matrix-coated surface.

[0056] In some embodiments, the matrix in the culture method may be laminin, vitrin, gelatin, polylysine, thromboretin, or Matrigel™. In some embodiments, the matrix in the culture method may be vitrin or Matrigel™. In some embodiments, the matrix in the culture method may contain DLL4, VCAM, and RetroNectin.

[0057] In this application, the culture method may include periodically or irregularly supplementing and / or replacing the culture medium.

[0058] In this application, the culture method may include digesting the cells into single cells after they have proliferated to a sufficient number.

[0059] In some embodiments, the digestion in the culture method uses Accutase digestion solution. In some embodiments, the digestion in the culture method uses Trypsin and EDTA.

[0060] In this application, the culture method may include culturing cells at a temperature of about 37-39°C, for example, about 36.5°C, about 37°C, about 37.5°C, about 38°C, about 38.5°C, about 39°C, or about 39.5°C.

[0061] In this application, the culture method may include culturing cells under conditions of about 3-7% CO2, for example, about 3% CO2, about 3.5% CO2, about 4% CO2, about 4.5% CO2, about 5% CO2, about 5.5% CO2, about 6% CO2, about 6.5% CO2, about 7% CO2, or about 7.5% CO2.

[0062] In this application, the culture method may include culturing cells under hypoxic conditions, which may include approximately 3-20% O2, for example, approximately 3% O2, approximately 3.5% O2, approximately 4% O2, approximately 4.5% O2, approximately 5% O2, approximately 5.5% O2, approximately 6% O2, approximately 6.5% O2, approximately 7% O2, approximately 7.5% O2, approximately 8% O2, approximately 8.5% O2, approximately 9% O2, approximately 9.5% O2, approximately 10% O2, approximately 10.5% O2, approximately 11% O2, approximately 11.5% O2, approximately 12% O2, approximately 12.5% ​​O2, approximately 13% O2, approximately 13.5% O2, approximately 14% O2, approximately 14.5% O2, approximately 15% O2, approximately 15.5% O2, approximately 16% O2, or approximately 16.5% O2. O2, approximately 17% O2, approximately 17.5% O2, approximately 18% O2, approximately 18.5% O2, approximately 19% O2, approximately 19.5% O2, approximately 20% O2, approximately 20.5% O2.

[0063] In this application, the culture method can be performed under serum-containing culture conditions. In this application, the culture method can also be performed under serum-free culture conditions.

[0064] In this application, the culture method can be performed under culture conditions without a feeder cell layer. In this application, the culture method can also be performed under culture conditions with a feeder cell layer.

[0065] In this application, the method can be an in vitro method. In this application, the method can be an ex vivo method.

[0066] In this application, the method may be a method for the purpose of non-disease diagnosis and treatment.

[0067] In this application, the sources of pluripotent cells, induced pluripotent stem cells, and hematopoietic stem / progenitor cells in the culture method are not limited. They can be of mammalian origin or non-mammal origin, and can be derived from human blood outside the body, umbilical cord blood, bone marrow, natural cells, or modified cells.

[0068] In this application, the method uses Rho activator I / II, which can activate Rho GTPase. Any natural or synthetic compound that can achieve the same function can be called Rho activator I / II. Rho activator I / II can convert glutamine on Rho GTPase to glutamate through deamination amidation. This chemical modification blocks the GTPase activity of Rho GTPase, thus keeping Rho GTPase in a normally active state. The main component of Rho activator I / II can be CN03 protein.

[0069] In this application, Rho activator I / II can be one or more of CN02, CN03 and CN04, and CN02, CN03 and CN04 can be mixed in different proportions, CN02 and CN03 can be mixed in different proportions, CN02 and CN04 can be mixed in different proportions, CN03 and CN04 can be mixed in different proportions, or CN02 can be a single component, CN03 can be a single component, or CN04 can be a single component.

[0070] In this application, the method uses Rho activator I / II, the concentration of which can be adaptively adjusted according to the culture conditions to achieve the best culture effect.

[0071] For example, the concentration can be 50-750 ng / mL, 50-700 ng / mL, 50-650 ng / mL, 50-600 ng / mL, 50-550 ng / mL, 50-500 ng / mL, 50-450 ng / mL, 50-400 ng / mL, 50-350 ng / mL, 50-300 ng / mL, 50-250 ng / mL, 50-200 ng / mL, 50-150 ng / mL, 50-100 ng / mL, 50-750 ng / mL, 100-750ng / mL, 100-700 ng / mL, 100-650 ng / mL, 100-600 ng / mL, 100-550 ng / mL, 100-500 ng / mL, 100-450 ng / mL, 100-400 ng / mL, 100-350 ng / mL, 100-300 ng / mL, 100-250 ng / mL, 100-200ng / mL, 100-150 ng / mL, 150-750 ng / mL, 150-700 ng / mL, 150-650 ng / mL, 150-600 ng / mL, 150-550 ng / mL, 150-500 ng / mL, 150-450 ng / mL, 150-400 ng / mL, 150-350 ng / mL, 150-300ng / mL, 150-250 ng / mL, 150-200 ng / mL, 200-750 ng / mL, 200-700 ng / mL, 200-650 ng / mL, 200-600 ng / mL, 200-550 ng / mL, 200-500 ng / mL, 200-450 ng / mL, 200-400 ng / mL, 200-350ng / mL, 200-300 ng / mL, 200-250 ng / mL, about 50 ng / mL, about 100 ng / mL, about 150 ng / mL, about 200ng / mL, about 250 ng / mL, about 300 ng / mL, about 350 ng / mL, about 400 ng / mL, about 450 ng / mL, about 500 ng / mL, about 550 ng / mL, about 600 ng / mL, about 650 ng / mL, about 700 ng / mL, approximately 750 ng / mL.

[0072] In this application, the time for culturing pluripotent cells using Rho activator I / II can be adaptively adjusted according to different cells and culture conditions to achieve the best culture results.

[0073] For example, the incubation time can be 12-96 hours, 12-84 hours, 12-72 hours, 12-60 hours, 12-48 hours, 12-36 hours, 12-24 hours, 24-96 hours, 24-84 hours, 24-72 hours, 24-60 hours, 24-48 hours, 24-36 hours, 36-96 hours, 36-84 hours, 36-72 hours, 36-60 hours, 36-48 hours, 48-96 hours, 48-84 hours, 48-72 hours, 48-60 hours, approximately 12 hours, approximately 24 hours, approximately 36 hours, approximately 48 hours, approximately 60 hours, approximately 72 hours, approximately 84 hours, and approximately 96 hours.

[0074] In this application, the method may use CN03, and the concentration of CN03 may be adaptively adjusted according to the culture conditions to achieve the best culture effect.

[0075] For example, the concentration can be 50-750 ng / mL, 50-700 ng / mL, 50-650 ng / mL, 50-600 ng / mL, 50-550 ng / mL, 50-500 ng / mL, 50-450 ng / mL, 50-400 ng / mL, 50-350 ng / mL, 50-300 ng / mL, 50-250 ng / mL, 50-200 ng / mL, 50-150 ng / mL, 50-100 ng / mL, 50-750 ng / mL, 100-750ng / mL, 100-700 ng / mL, 100-650 ng / mL, 100-600 ng / mL, 100-550 ng / mL, 100-500 ng / mL, 100-450 ng / mL, 100-400 ng / mL, 100-350 ng / mL, 100-300 ng / mL, 100-250 ng / mL, 100-200ng / mL, 100-150 ng / mL, 150-750 ng / mL, 150-700 ng / mL, 150-650 ng / mL, 150-600 ng / mL, 150-550 ng / mL, 150-500 ng / mL, 150-450 ng / mL, 150-400 ng / mL, 150-350 ng / mL, 150-300ng / mL, 150-250 ng / mL, 150-200 ng / mL, 200-750 ng / mL, 200-700 ng / mL, 200-650 ng / mL, 200-600 ng / mL, 200-550 ng / mL, 200-500 ng / mL, 200-450 ng / mL, 200-400 ng / mL, 200-350ng / mL, 200-300 ng / mL, 200-250 ng / mL, about 50 ng / mL, about 100 ng / mL, about 150 ng / mL, about 200ng / mL, about 250 ng / mL, about 300 ng / mL, about 350 ng / mL, about 400 ng / mL, about 450 ng / mL, about 500 ng / mL, about 550 ng / mL, about 600 ng / mL, about 650 ng / mL, about 700 ng / mL, approximately 750 ng / mL.

[0076] In this application, the time for culturing pluripotent cells using CN03 can be adaptively adjusted according to different cells and culture conditions to achieve the best culture results.

[0077] For example, the incubation time can be 12-96 hours, 12-84 hours, 12-72 hours, 12-60 hours, 12-48 hours, 12-36 hours, 12-24 hours, 24-96 hours, 24-84 hours, 24-72 hours, 24-60 hours, 24-48 hours, 24-36 hours, 36-96 hours, 36-84 hours, 36-72 hours, 36-60 hours, 36-48 hours, 48-96 hours, 48-84 hours, 48-72 hours, 48-60 hours, approximately 12 hours, approximately 24 hours, approximately 36 hours, approximately 48 hours, approximately 60 hours, approximately 72 hours, approximately 84 hours, and approximately 96 hours.

[0078] In this application, the T cells include T cells with different phenotypes, which can be modified T cells or unmodified T cells. For example, the T cells can be thymocytes, naive T lymphocytes, immature T lymphocytes, mature T lymphocytes, resting T lymphocytes, activated T lymphocytes, and helper T cells (HTL; CD4). + T cells, cytotoxic T cells (CTL; ​​CD8) + T cells, CD4 + CD8 + T cells, CD4 - CD8 - T cells, and any other subsets of T cells. For example, the T cells can be CAR-T cells and TCR-T cells. For example, the T cells can be CD5+CD7+ T cell precursors or CD5+CD8αβ+ T cells.

[0079] In this application, the method includes culturing pluripotent cells using a culture medium.

[0080] In this application, the culture medium may comprise a basal culture medium. For example, the basal culture medium may comprise any culture medium known in the art. For example, the basal culture medium may comprise one or more of IMDM, MEM, Ham's F12, mTeSR1, APEL, StemSpan™ SFEM II, DMEM, Nuwacell® ncTarget complete medium, Stempro-34 complete medium, and RPMI 1640. For example, the basal culture medium may be Nuwacell® ncTarget complete medium. For example, the basal culture medium may be StemSpan™ SFEM II. For example, the basal culture medium may be Stempro-34 complete medium.

[0081] In this application, the culture medium may contain one or more of nutrients, extracts, growth factors, hormones, cytokines, and culture medium additives. The types and concentrations of the added components can be adaptively adjusted according to the culture conditions to achieve better culture results.

[0082] For example, one or more of the following components may be added: serum substitutes, glutamine, NEAA (non-essential amino acids), ascorbic acid, epidermal growth factor (EGF), acidic fibroblast growth factor (aFGF), basic fibroblast growth factor (bFGF), leukemia inhibitory factor (LIF), hepatocyte growth factor (HGF), insulin-like growth factor 1 (IGF-1), insulin-like growth factor 2 (IGF-2), keratinocyte growth factor (KGF), nerve growth factor (NGF), platelet-derived growth factor (PDGF), transforming growth factor β (TGF-β), bone morphogenetic protein (BMP4), and vascular endothelial growth factor. The additives include: VEGF, transferrin, insulin, selenium, various interleukins (e.g., IL-1 to IL-18), various colony-stimulating factors (e.g., granulocyte / macrophage colony-stimulating factor (GM-CSF)), various interferons (e.g., IFN-γ), stem cell factor (SCF), thrombopoietin (TPO), erythropoietin (EPO), N2 additive, B27 additive, and Fms-associated tyrosine kinase 3 ligand (FLt3). These additives are not limited to any particular source and may be commercially available, natural, or recombinant.

[0083] For example, one or more of the following ingredients may be added: CHIR99021, Y-27632, GlutaMAX, L-ascorbic acid, MTG, ITS, BMP4, VEGF, bFGF, SB 431542, SCF, TPO, FLT3, Sodium Pyruvate, β-Mercaptoethanol, NEAA, IL-7, IL-15, SB203580, SDF-1α, Dexamethasone, IL-21, and Anti-CD3 (OKT3).

[0084] Culture medium

[0085] On the other hand, this application provides a culture medium for culturing pluripotent cells and differentiating them into T cells, the culture medium containing Rho activator I / II.

[0086] In this application, Rho activator II can activate Rho GTPase. Natural or synthetic compounds that can achieve the same function can be called Rho activators I / II. Rho activators I / II can convert glutamine on Rho GTPase to glutamate through deamination amidation. This chemical modification blocks the GTPase activity of Rho GTPase, thus keeping Rho GTPase in a normally active state. The main component of Rho activator I / II can be CN03 protein.

[0087] In this application, Rho activator I / II can be one or more of CN02, CN03 and CN04, and CN02, CN03 and CN04 can be mixed in different proportions, CN02 and CN03 can be mixed in different proportions, CN02 and CN04 can be mixed in different proportions, CN03 and CN04 can be mixed in different proportions, or CN02 can be a single component, CN03 can be a single component, or CN04 can be a single component.

[0088] In this application, the concentration of Rho activator I / II can be adaptively adjusted according to the culture conditions to achieve the best culture effect.

[0089] For example, the concentration can be 50-750 ng / mL, 50-700 ng / mL, 50-650 ng / mL, 50-600 ng / mL, 50-550 ng / mL, 50-500 ng / mL, 50-450 ng / mL, 50-400 ng / mL, 50-350 ng / mL, 50-300 ng / mL, 50-250 ng / mL, 50-200 ng / mL, 50-150 ng / mL, 50-100 ng / mL, 50-750 ng / mL, 100-750ng / mL, 100-700 ng / mL, 100-650 ng / mL, 100-600 ng / mL, 100-550 ng / mL, 100-500 ng / mL, 100-450 ng / mL, 100-400 ng / mL, 100-350 ng / mL, 100-300 ng / mL, 100-250 ng / mL, 100-200ng / mL, 100-150 ng / mL, 150-750 ng / mL, 150-700 ng / mL, 150-650 ng / mL, 150-600 ng / mL, 150-550 ng / mL, 150-500 ng / mL, 150-450 ng / mL, 150-400 ng / mL, 150-350 ng / mL, 150-300ng / mL, 150-250 ng / mL, 150-200 ng / mL, 200-750 ng / mL, 200-700 ng / mL, 200-650 ng / mL, 200-600 ng / mL, 200-550 ng / mL, 200-500 ng / mL, 200-450 ng / mL, 200-400 ng / mL, 200-350ng / mL, 200-300 ng / mL, 200-250 ng / mL, about 50 ng / mL, about 100 ng / mL, about 150 ng / mL, about 200ng / mL, about 250 ng / mL, about 300 ng / mL, about 350 ng / mL, about 400 ng / mL, about 450 ng / mL, about 500 ng / mL, about 550 ng / mL, about 600 ng / mL, about 650 ng / mL, about 700 ng / mL, approximately 750 ng / mL.

[0090] In this application, the Rho activator I / II can be CN03, and the concentration of CN03 can be adaptively adjusted according to the culture conditions to achieve the best culture effect.

[0091] For example, the concentration can be 50-750 ng / mL, 50-700 ng / mL, 50-650 ng / mL, 50-600 ng / mL, 50-550 ng / mL, 50-500 ng / mL, 50-450 ng / mL, 50-400 ng / mL, 50-350 ng / mL, 50-300 ng / mL, 50-250 ng / mL, 50-200 ng / mL, 50-150 ng / mL, 50-100 ng / mL, 50-750 ng / mL, 100-750 ng / mL, 100-700 ng / mL, 100-650 ng / mL, 100-600 ng / mL, 100-550 ng / mL, 100-500 ng / mL, 100-450 ng / mL, 100-400 ng / mL, 100-350 ng / mL, 100-300 ng / mL, 100-250 ng / mL, 100-200ng / mL, 100-150 ng / mL, 150-750 ng / mL, 150-700 ng / mL, 150-650 ng / mL, 150-600 ng / mL, 150-550 ng / mL, 150-500 ng / mL, 150-450 ng / mL, 150-400 ng / mL, 150-350 ng / mL, 150-300ng / mL, 150-250 ng / mL, 150-200 ng / mL, 200-750 ng / mL, 200-700 ng / mL, 200-650 ng / mL, 200-600 ng / mL, 200-550 ng / mL, 200-500 ng / mL, 200-450 ng / mL, 200-400 ng / mL, 200-350ng / mL, 200-300 ng / mL, 200-250 ng / mL, about 50 ng / mL, about 100 ng / mL, about 150 ng / mL, about 200 ng / mL, about 250 ng / mL, about 300 ng / mL, about 350 ng / mL, about 400 ng / mL, about 450 ng / mL, about 500 ng / mL, about 550 ng / mL, about 600 ng / mL, about 650 ng / mL, about 700 ng / mL, about 750 ng / mL.

[0092] In this application, the culture medium may comprise a basal culture medium. For example, the basal culture medium may comprise any culture medium known in the art. For example, the basal culture medium may comprise one or more of IMDM, MEM, Ham's F12, mTeSR1, APEL, StemSpan™ SFEM II, DMEM, Nuwacell® ncTarget complete medium, Stempro-34 complete medium, and RPMI 1640. For example, the basal culture medium may be Nuwacell® ncTarget complete medium. For example, the basal culture medium may be StemSpan™ SFEM II. For example, the basal culture medium may be Stempro-34 complete medium.

[0093] In this application, the culture medium may contain one or more of nutrients, extracts, growth factors, hormones, cytokines, and culture medium additives. The types and concentrations of the added components can be adaptively adjusted according to the culture conditions to achieve better culture results.

[0094] For example, one or more of the following components may be added: serum substitutes, glutamine, NEAA (non-essential amino acids), ascorbic acid, epidermal growth factor (EGF), acidic fibroblast growth factor (aFGF), basic fibroblast growth factor (bFGF), leukemia inhibitory factor (LIF), hepatocyte growth factor (HGF), insulin-like growth factor 1 (IGF-1), insulin-like growth factor 2 (IGF-2), keratinocyte growth factor (KGF), nerve growth factor (NGF), platelet-derived growth factor (PDGF), transforming growth factor β (TGF-β), bone morphogenetic protein (BMP4), and vascular endothelial growth factor. The additives include: VEGF, transferrin, insulin, selenium, various interleukins (e.g., IL-1 to IL-18), various colony-stimulating factors (e.g., granulocyte / macrophage colony-stimulating factor (GM-CSF)), various interferons (e.g., IFN-γ), stem cell factor (SCF), thrombopoietin (TPO), erythropoietin (EPO), N2 additive, B27 additive, and Fms-associated tyrosine kinase 3 ligand (FLT3). These additives are not limited to any particular source and may be commercially available, natural, or recombinant.

[0095] For example, one or more of the following ingredients may be added: CHIR99021, Y-27632, GlutaMAX, L-ascorbic acid, MTG, ITS, BMP4, VEGF, bFGF, SB 431542, SCF, TPO, FLT3, Sodium Pyruvate, β-Mercaptoethanol, NEAA, IL-7, IL-15, SB203580, SDF-1α, Dexamethasone, and Anti-CD3 (OKT3).

[0096] Compositions, uses

[0097] On the other hand, this application provides a composition comprising pluripotent cells and the culture medium described in this application.

[0098] On the other hand, this application provides a composition comprising induced pluripotent stem cells and the culture medium described in this application.

[0099] On the other hand, this application provides a composition comprising hematopoietic stem / progenitor cells and the culture medium described in this application.

[0100] On the other hand, this application also provides a culture platform for obtaining T cells, which includes the method and the culture medium.

[0101] On the other hand, this application also provides a method for preventing and / or treating a disease, comprising administering T cells to a subject in need, the T cells being obtained by using the method, the culture medium, the composition, and the culture platform.

[0102] For example, the administered T cells can be pharmaceutically formulated according to any conventional method. For instance, carriers, excipients, or diluents can be used to mix or dilute the active ingredient. Examples of suitable carriers, excipients, or diluents are lactose, dextrose, sucrose, sorbitol, mannitol, glycine, polyethylene glycol, starch, gum arabic, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylparaben, propylparaben, talc, magnesium stearate, and mineral oil. The formulation may additionally include, for example, fillers, anticoagulants, lubricants, wetting agents, flavoring agents, emulsifiers, preservatives, etc. The compositions of the present invention can be formulated using any method known in the art to provide a rapid, sustained, or delayed release of the active ingredient upon administration to a patient.

[0103] The cells in this application can be administered by injection (e.g., intramuscular, intravenous, intraperitoneal, subcutaneous) or by other methods such as infusion to ensure they enter the bloodstream in an effective form. The cells can also be administered via intratumoral, peritumoral, intralesional, or perilesional routes to exert local and systemic therapeutic effects. For example, they can be administered via local or intravenous injection.

[0104] In this application, the dosage of the cells can be a single dose or multiple doses. For example, the actual dosage of the cells can be determined based on a variety of relevant factors, such as the type of disease; the route of administration; the patient's age, sex, and / or weight; and the severity of the patient's symptoms.

[0105] This application also provides the following implementation methods:

[0106] 1. A method for proliferating and / or differentiating T cells, comprising culturing pluripotent cells using Rho activator I / II and proliferating and / or differentiating the pluripotent cells into T cells.

[0107] 2. The method according to Embodiment 1, wherein the pluripotent cell is an induced pluripotent stem cell (iPSC).

[0108] 3. The method according to Embodiment 1, wherein the pluripotent cell is a hematopoietic stem / progenitor cell (HPC).

[0109] 4. The method according to any one of embodiments 1-3, wherein the pluripotent cell is a modified cell.

[0110] 5. The method according to any one of embodiments 1-4, wherein the Rho activator I / II is selected from one or more activators of the group consisting of CN02, CN03 and CN04.

[0111] 6. The method according to any one of embodiments 1-5, wherein the Rho activator II is CN02.

[0112] 7. The method according to any one of embodiments 1-6, wherein the Rho activator II is CN03.

[0113] 8. The method according to any one of embodiments 1-7, wherein the Rho activator I is CN04.

[0114] 9. The method according to any one of embodiments 1-8, wherein the method comprises culturing pluripotent cells using Rho activator I / II for 12-96 hours.

[0115] 10. The method according to any one of embodiments 1-9, wherein the method comprises culturing pluripotent cells using Rho activator I / II for 12-72 hours.

[0116] 11. The method according to any one of embodiments 1-10, wherein the method comprises culturing pluripotent cells using Rho activator I / II for 12-48 hours.

[0117] 12. The method according to any one of embodiments 1-11, wherein the method comprises culturing pluripotent cells using Rho activator I / II for 24-48 hours.

[0118] 13. The method according to any one of embodiments 1-12, wherein the method comprises culturing pluripotent cells using CN03 for approximately 48 hours.

[0119] 14. The method according to any one of embodiments 11-13, wherein the concentration of the Rho activator I / II is 50-750 ng / mL.

[0120] 15. The method according to any one of embodiments 1-14, wherein the concentration of the Rho activator I / II is 150-350 ng / mL.

[0121] 16. The method according to any one of embodiments 1-15, wherein the concentration of the Rho activator I / II is about 250 ng / mL.

[0122] 17. The method according to any one of embodiments 5-16, wherein the concentration of CNO3 is 50-750 ng / mL.

[0123] 18. The method according to any one of embodiments 5-17, wherein the concentration of CNO3 is 150-350 ng / mL.

[0124] 19. The method according to any one of embodiments 5-18, wherein the concentration of CNO3 is about 250 ng / mL.

[0125] 20. The method according to any one of embodiments 1-19, wherein the T cells are CD5+CD7+ T precursor cells.

[0126] 21. The method according to any one of embodiments 1-20, wherein the T cells are CD5+CD8αβ+ T cells.

[0127] 22. The method according to any one of embodiments 1-21, wherein the method comprises culturing cells under hypoxic conditions.

[0128] 23. The method according to any one of embodiments 1-22, wherein the low-oxygen condition is 5% O2.

[0129] 24. The method according to any one of embodiments 1-23, wherein the method comprises culturing cells under serum-free culture conditions.

[0130] 25. The method according to any one of embodiments 1-24, wherein the method does not use trophoblast cells.

[0131] 26. The method according to any one of embodiments 1-25, wherein the method comprises culturing pluripotent cells using a culture medium containing one or more of the following factors: CHIR99021, Y-27632, GlutaMAX, L-ascorbic acid, MTG, ITS, BMP4, VEGF, bFGF, SB 431542, SCF, TPO, FLT3, Sodium Pyruvate, β-Mercaptoethanol, NEAA, IL-7, IL-15, SB203580, SDF-1α, Dexamethasone, IL-21, and Anti-CD3 OKT3.

[0132] 27. The method according to any one of embodiments 1-26, wherein the method comprises culturing pluripotent cells using a culture medium comprising a basal culture medium.

[0133] 28. The method according to any one of embodiments 1-27, wherein the basal culture medium is selected from one or more of the following culture media: Nuwacell® ncTarget complete medium, Stempro-34 complete medium and StemSpan SFEM II medium.

[0134] 29. A culture medium for culturing pluripotent cells and differentiating them into T cells, said culture medium containing Rho activator I / II.

[0135] 30. The culture medium according to embodiment 29, wherein the pluripotent cells are induced pluripotent stem cells (iPSCs).

[0136] 31. The culture medium according to Embodiment 29, wherein the pluripotent cells are hematopoietic stem / progenitor cells (HPCs).

[0137] 32. The culture medium according to any one of embodiments 29-31, wherein the pluripotent cells are modified cells.

[0138] 33. The culture medium according to any one of embodiments 29-32, wherein the Rho activator I / II is selected from one or more activators of the group consisting of CN02, CN03 and CN04.

[0139] 34. The culture medium according to any one of embodiments 29-33, wherein the Rho activator II is CN02.

[0140] 35. The culture medium according to any one of embodiments 29-34, wherein the Rho activator II is CN03.

[0141] 36. The culture medium according to any one of embodiments 29-35, wherein the Rho activator I is CN04.

[0142] 37. The culture medium according to any one of embodiments 29-36, wherein the concentration of Rho activator I / II is 50-750 ng / mL.

[0143] 38. The culture medium according to any one of embodiments 29-37, wherein the concentration of Rho activator I / II is 150-350 ng / mL.

[0144] 39. The culture medium according to any one of embodiments 29-38, wherein the concentration of Rho activator I / II is about 250 ng / mL.

[0145] 40. The culture medium according to any one of embodiments 33-39, wherein the concentration of CNO3 is 50-750 ng / mL.

[0146] 41. The culture medium according to any one of embodiments 33-40, wherein the concentration of CNO3 is 150-350 ng / mL.

[0147] 42. The culture medium according to any one of embodiments 33-41, wherein the concentration of CNO3 is about 250 ng / mL.

[0148] 43. The culture medium according to any one of embodiments 29-42, wherein the T cells are CD5+CD7+ T precursor cells.

[0149] 44. The culture medium according to any one of embodiments 29-43, wherein the T cells are CD5+CD8αβ+ T cells.

[0150] 45. The culture medium according to any one of embodiments 29-44, comprising one or more of the following factors: CHIR99021, Y-27632, GlutaMAX, L-ascorbic acid, MTG, ITS, BMP4, VEGF, bFGF, SB431542, SCF, TPO, FLT3, Sodium Pyruvate, β-Mercaptoethanol, NEAA, IL-7, IL-15, SB203580, SDF-1α, Dexamethasone, IL-21, and Anti-CD3 OKT3.

[0151] 46. ​​The culture medium according to any one of embodiments 29-45, comprising a basal culture medium.

[0152] 47. The culture medium according to any one of embodiments 29-46, wherein the basal culture medium is selected from one or more of the following culture media: Nuwacell® ncTarget complete medium, Stempro-34 complete medium and StemSpan SFEM II medium.

[0153] 48. A composition comprising pluripotent cells and a culture medium according to any one of embodiments 29-47.

[0154] 49. A culture platform for obtaining T cells, comprising the method of any one of embodiments 1-28 and / or the culture medium of any one of embodiments 29-47.

[0155] 50. Application of the culture platform described in Embodiment 49, comprising the method of any one of Embodiments 1-28 and / or the culture medium of any one of Embodiments 29-47.

[0156] The embodiments described below are not intended to be limited by any theory, but are merely for illustrating the preparation method and uses of this application, and are not intended to limit the scope of the invention.

[0157] Example

[0158] Example 1: A 3D differentiation method for inducing iPSCs to generate hematopoietic stem / progenitor cells

[0159] 1) When the iPSCs reach approximately 80% confluence, transfer them from the incubator to the biosafety cabinet and discard the culture medium. Rinse the bottom of the culture flask with DPBS (calcium and magnesium ions-free) and then discard the DPBS. Add an appropriate amount of Accutase (1 mL / 10 cm²). 2 Add the solution to a six-well plate and shake to thoroughly wet the bottom. Then transfer the six-well plate to a 37°C incubator for digestion.

[0160] 2) After digestion until the cells detach from the bottom of the culture plate, terminate the digestion with Nuwacell® ncTarget medium. After centrifugation, remove the supernatant, resuspend the cells thoroughly in Nuwacell® ncTarget medium, and filter the cells through a 40 μM sieve. AO / PI sampling and counting are performed. Seed the cells at a density of 0.6 x 10e6 / mL into low-absorption six-well plates (small system) or conical flasks (large system). Transfer the six-well plates to a horizontal shaker in a hypoxic incubator (5% O2) at 80 rpm. This is day 0 of iPSC-HPC differentiation.

[0161] Table 1. Culture medium composition (D0)

[0162]

[0163] 3) On differentiation day D1, let the 6-well plate stand for 5 minutes to allow the EB bulbs to settle to the bottom. Gently aspirate the supernatant (at least 85% of the total volume) into a 15 mL centrifuge tube and centrifuge. After centrifugation, remove the supernatant and resuspend the cells in 2 mL of differentiation medium (D1). Transfer the cell suspension to the 6-well plate. Transfer the 6-well plate to a horizontal shaker in a hypoxic incubator (5% O2) at a shaking speed of 80 rpm.

[0164] Table 2. Culture medium composition (D1)

[0165]

[0166] 4) On day 2 of differentiation, let the six-well plate stand for 5 minutes until the EB pellets sink to the bottom. Gently aspirate 85% of the total supernatant and add fresh differentiation medium (D2-D3) to the six-well plate. Transfer the six-well plate to a horizontal shaker in a hypoxic incubator and incubate at 80 rpm for 24 hours.

[0167] 5) On day 3 of differentiation, let the 6-well plate stand for 5 minutes until the EB pellets sink to the bottom. Gently aspirate 85% of the total supernatant and add fresh differentiation medium (D2-D3) to the 6-well plate. Transfer the 6-well plate to a horizontal shaker in a hypoxic incubator and incubate at 80 rpm for 24 hours.

[0168] Table 3. Culture medium composition (D2-D3)

[0169]

[0170] 6) On day 4 of differentiation, let the 6-well plate stand for 5 minutes to allow the EB pellets to settle to the bottom. Gently aspirate 85% of the total supernatant (if suspended cells are present, the supernatant needs to be removed after centrifugation), and add fresh differentiation medium (D4-D6) to the 6-well plate. Transfer the 6-well plate to a horizontal shaker in an ambient oxygen incubator at 80 rpm. Incubate for 24 hours. Change the medium once daily, following the same steps as above.

[0171] Table 4. Culture medium composition (D4-D6)

[0172]

[0173] 7) On days 7-14 of differentiation, let the 6-well plate stand for 5 minutes to allow the EB pellets to settle to the bottom. Gently aspirate 85% of the total supernatant (if suspended cells are present, the supernatant needs to be removed after centrifugation), and add fresh differentiation medium (D7-D14) to the 6-well plate. Transfer the 6-well plate to a horizontal shaker in an ambient oxygen incubator at 80 rpm. Incubate for 24 hours. Change the medium daily as above.

[0174] Table 5. Culture medium composition (D7-D14)

[0175]

[0176] 8) On day 14 of differentiation, suspension cells were collected and HPC purity was determined by flow cytometry.

[0177] The above experiments show that:

[0178] As shown in Figure 1, the method described in Example 1 requires no serum or feeder cells throughout the entire process. iPSCs are cultured in suspension in conical flasks / six-well plates on a shaker at 80 rpm, allowing them to spontaneously form EB spheres. The spheres are uniform in size, the operation is simple, and it is easy to scale up for large-scale use. The generated hematopoietic stem cells are gradually released into the suspension system, making them easy to collect without the need for EB sphere digestion, and the expansion fold is between 5 and 10 times. The generated CD45+CD34+CD38-CD235a- cells have high purity and can be directly used for subsequent cell differentiation or library construction without the need for flow cytometry or magnetic bead sorting.

[0179] Example 2: Inducing iPSCs to generate hematopoietic stem / progenitor cells by adding small molecule compounds

[0180] To test the function of small molecule additives in iPSC-HPC differentiation, different concentrations of CN03 and Endothelin-1 were added at different time points from D5 to D14 in the iPSC differentiation method used in Example 1 to test the HPC yield.

[0181] 1) CN03 was added at a concentration of 250 ng / mL to differentiated cells D5-D7, D7-D9, and D5-D9, respectively. Furthermore, it was added to differentiated cells D5-D7 at concentrations of 50 ng / mL, 150 ng / mL, 250 ng / mL, 350 ng / mL, 750 ng / mL, and 1000 ng / mL, respectively, to test the phenotype and yield of HPC.

[0182] 2) Endothelin-1 was added at concentrations of 10 ng / mL and 100 ng / mL during differentiation at D5-D9 and D8-D14, respectively.

[0183] The above experiments show that:

[0184] 1) CNO3: As shown in Figure 2, the addition of 250 ng / mL CNO3 on Day 5-Day 7 significantly increased the HPC yield (approximately 4-fold increase), and on Day 7-Day 9, it increased the HPC yield (approximately 2-fold increase), but had no effect on the yield on Day 5-9. When adding different concentrations of CNO3 on Day 5-Day 7, 150 ng / mL-350 ng / mL all increased the yield, with 250 ng / mL CNO3 showing the highest yield increase of approximately 4-5 times compared to the control group. 50 ng / mL did not significantly increase the yield, while 750 ng / mL and 1000 ng / mL significantly decreased the yield and affected the phenotype. Therefore, adding 250 ng / mL CNO3 on Day 5-Day 7 maximizes the HPC yield.

[0185] 2) Endothelin-1: As shown in Figure 2, when Endothelin-1 was added at concentrations of 100 ng / mL and 10 ng / mL on Days 5-9 and 8-14, it only increased the HPC yield by a maximum of about 1.5 times.

[0186] Example 3: HPCs generated by iPSCs differentiate into T cells

[0187] 1) Early T cell differentiation (D0-D14): Prepare PBS-diluted "DVR" coating medium (8 μg / mL VCAM-1 + 20 μg / mL DLL4 + 10 μg / mL RetroNectine), and coat at room temperature for 2 hours or overnight at 4°C. Take suspension cells from Examples 1 and 2 on day 14 of differentiation, seed approximately 10,000 HPCs per well in a 24-well plate, and add T cell differentiation medium. Add fresh medium after three days, and then perform half-medium medium replacement every three days thereafter. On day 14 of differentiation, collect cells into 15 mL centrifuge tubes, wash once with PBS, centrifuge at room temperature, discard the supernatant, resuspend in an appropriate amount of differentiation medium, gently mix, and then perform cell counting by mixing the cell suspension with AO / PI staining solution at a 1:1 ratio. Take an appropriate amount of cell suspension for flow cytometry phenotypic analysis of the proportion and phenotype of T cell precursors (CD5, CD7, CD8a, CD56).

[0188] Table 6. Composition of T cell differentiation culture medium

[0189]

[0190] 2) Late T cell differentiation (D14-D21): Prepare a solution containing 1 μg / mL DLL4 and 10 μg / mL RetroNectine in PBS to coat cell culture plates (coat at room temperature for 2 hours or overnight at 4°C, then dilute with PBS). Seed 500,000 T cells on day 14 of differentiation into each well with T differentiation medium. After 3 days, add twice the amount of T differentiation medium to each well, and then perform half-medium medium replacement every 3 days thereafter. On day 21 of differentiation: collect cells into 15 mL centrifuge tubes, centrifuge at room temperature, discard the supernatant, resuspend in medium, gently mix, and then perform cell counting with 20 μl of cell suspension and 20 μl of AO / PI staining solution. Perform flow cytometry phenotypic analysis on T cell expression.

[0191] 3) T cell maturation: Prepare a solution containing 1 μg / mL DLL4 and 10 μg / mL RetroNectine using PBS, coat the cell culture plate, and incubate at room temperature for 2 hours or overnight at 4°C. Wash once with PBS before use. T cells on day 21 of differentiation are resuspended in Maturation medium 1 and transferred to wells, and incubated at 37°C for 3 days. Prepare RetroNectine dilution buffer using PBS, coat the culture plate at room temperature for 2 hours or overnight at 4°C, and wash once with PBS before use. Collect cells on day 3 of maturation, centrifuge to remove the supernatant, resuspend in Maturation medium 2, and transfer to wells coated with RetroNectine. After incubation at 37°C for 4 days, collect cells, and take an appropriate amount of cell suspension for flow cytometry phenotypic analysis of CD4 / CD8a / CD8b / CD5.

[0192] Table 7. Components of Maturation Culture Medium

[0193]

[0194] 4) Amplification capacity test: HPCs obtained using the iPSCs optimization method listed in Examples 2 and 3 were further differentiated into T cells and induced to mature. After two rounds of amplification using anti-CD3 antibody, an amplification of approximately 400,000 times from iPSCs to T cells could be achieved.

[0195] The above experimental results show that:

[0196] 1) As shown in Figures 4 and 5, iPSCs obtained through the 3D differentiation methods listed in Examples 1 and 2 can efficiently generate CD5+CD7+ T precursor cells and CD5+CD8αβ+ T cells with typical characteristics. The entire process requires no serum or feeder cells, making it safe and controllable.

[0197] 2) As shown in Figure 4, the addition of the small molecule compound CN03 (250 ng / mL, Day 5-7) during the iPSC to HPCs generation stage increased the proportion of CD5+CD7+ T cell precursors in the early differentiation process of the generated HPCs and significantly improved the yield of T cell precursor cells.

[0198] 3) As shown in Figure 5, during the stage of iPSCs generating HPCs, the addition of CN03 on Day 5-Day 7 significantly increased the yield of HPCs subsequently generating mature iT cells. However, the addition of the small molecule compound Endothelin-1 had no effect.

[0199] 4) such as Figure 6 As shown, the iT cells obtained by the differentiation method used in this patent can be expanded with high efficiency. Finally, each iT cells generated from iPSCs can achieve an expansion efficiency of about 400,000 times after two rounds of expansion .

[0200] Example 4: HPCs generated by iPSCs differentiate into NK cells

[0201] In Example 1, on day 14 of iHPC differentiation, all suspension cells and EB spheres were collected by centrifugation. The cells were resuspended in NK differentiation medium 1 (as listed below) to a density of 2 million / mL. The culture vessel was transferred to a horizontal shaker in a 5% CO2 incubator at 80 rpm. The supernatant was removed and fresh medium was added every two days. For the first 7 days, NK differentiation medium 1 was used, and the cell density was adjusted to 2 million / mL. For the next 7 days, NK differentiation medium 2 was used, and the cell density was adjusted to 3 million / mL. On day 14 of NK differentiation, all cells were collected, counted, and analyzed by flow cytometry to detect CD94 / CD56 / CD16 / NKG2A.

[0202] Table 8. NK differentiation medium 1

[0203]

[0204] Table 9. NK differentiation medium 2

[0205]

[0206] The above experimental results show that:

[0207] As shown in Figure 7, the method for differentiating iPSCs into HPCs listed in Example 1 can further differentiate them into NK cells with high efficiency in a feederless, suspension culture system, with a ratio as high as 50%-80%, and each iPSC cell can differentiate into 5,000-10,000 NK cells.

Claims

1. A method for proliferating and / or differentiating T cells, comprising culturing pluripotent cells using Rho activator I / II and proliferating and / or differentiating the pluripotent cells into T cells.

2. The method according to claim 1, wherein the Rho activator I / II is selected from one or more activators of the group consisting of CN02, CN03 and CN04.

3. The method according to any one of claims 1-2, wherein the method comprises culturing pluripotent cells using Rho activator I / II for 24-48 hours.

4. The method according to any one of claims 1-3, wherein the concentration of the Rho activator I / II is 150-350 ng / mL.

5. The method according to any one of claims 1-4, wherein the method comprises culturing cells under hypoxic conditions.

6. The method according to any one of claims 1-5, wherein the method comprises culturing pluripotent cells using a culture medium containing one or more of the following factors: CHIR99021, Y-27632, GlutaMAX, L-ascorbic acid, MTG, ITS, BMP4, VEGF, bFGF, SB 431542, SCF, TPO, FLT3, Sodium Pyruvate, β-Mercaptoethanol, NEAA, IL-7, IL-15, SB203580, SDF-1α, Dexamethasone, IL-21, and Anti-CD3 OKT3.

7. A culture medium for culturing pluripotent cells and differentiating them into T cells, said culture medium containing Rho activator I / II.

8. The culture medium according to claim 7, wherein the Rho activator I / II is selected from one or more activators of the group consisting of CN02, CN03 and CN04.

9. The culture medium according to any one of claims 7-8, wherein the concentration of the Rho activator I / II is 150-350 ng / mL.

10. The culture medium according to any one of claims 7-9, comprising one or more of the following factors: CHIR99021, Y-27632, GlutaMAX, L-ascorbic acid, MTG, ITS, BMP4, VEGF, bFGF, SB 431542, SCF, TPO, FLT3, Sodium Pyruvate, β-Mercaptoethanol, NEAA, IL-7, IL-15, SB203580, SDF-1α, Dexamethasone, IL-21, and Anti-CD3 OKT3.

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