Culture composition and culture system

Abstract

The invention discloses a culture composition. According to the embodiment of the invention, the culture composition comprises a small molecule drug and a cell factor, and the small molecule drug comprises at least one of LBH589, LY2090314 and Selumetinib; the cell factors comprise at least one of IL-6 (interleukin-6), IL-3 (interleukin-3), IL-15 (interleukin-15), IL-2 (interleukin-2), SCF (mesenchymal stem cells) and Flt3L According to the culture composition provided by the embodiment of the invention, the efficiency of reprogramming the peripheral blood mononuclear cells into the induced pluripotent stem cells can be improved.

Description

Technical Field

[0001] This invention belongs to the field of cell culture. Specifically, this invention relates to a culture composition, and more specifically, to a culture system, a method for reprogramming peripheral blood mononuclear cells, a method for detecting the reprogramming efficiency of peripheral blood mononuclear cells, an induced pluripotent stem cell, and the application of induced pluripotent stem cells in regenerative medicine. Background Technology

[0002] Induced pluripotent stem cells (iPSCs) are pluripotent stem cells with characteristics similar to embryonic stem cells, obtained by reprogramming somatic cells. iPSCs have broad application prospects in clinical treatment and drug development, but current methods for reprogramming peripheral blood mononuclear cells (PBMCs) into iPSCs suffer from low efficiency and complex operation.

[0003] Therefore, there is an urgent need to develop a method for reprogramming peripheral blood mononuclear cells into induced pluripotent stem cells (iPSCs) to improve the efficiency of PBMC reprogramming into iPSCs. Summary of the Invention

[0004] The present invention aims to at least partially solve at least one of the technical problems existing in the prior art. To this end, the present invention provides a culture composition.

[0005] This invention is based on the following discoveries of the inventors:

[0006] Peripheral blood mononuclear cells (PBMCs) are widely used as a source for cell reprogramming. Obtaining iPSCs from PBMCs has the advantages of minimal skin invasiveness and easy acquisition. However, existing methods for reprogramming PBMCs into induced pluripotent stem cells (iPSCs) are time-consuming, cumbersome, costly, and have low efficiency in obtaining iPSCs. To overcome this problem, the inventors have proposed a culture composition that can improve the efficiency of PBMC reprogramming into iPSCs.

[0007] In a first aspect, the present invention provides a culture composition. According to embodiments of the invention, the culture composition comprises: a small molecule drug and cytokines, wherein the small molecule drug comprises at least one of LBH589, LY2090314, and Selumetinib; and the cytokines comprise at least one of IL-6, IL-3, IL-15, IL-2, SCF, and Flt3L. The culture composition according to embodiments of the invention can improve the efficiency of reprogramming peripheral blood mononuclear cells into induced pluripotent stem cells.

[0008] In a second aspect, the present invention provides a culture system. According to an embodiment of the invention, the culture system comprises a culture medium and the culture composition described in the first aspect of the invention. The culture system according to an embodiment of the invention can improve the efficiency of reprogramming peripheral blood mononuclear cells into induced pluripotent stem cells.

[0009] In a third aspect, the present invention provides a method for reprogramming peripheral blood mononuclear cells. According to embodiments of the invention, the method includes culturing peripheral blood mononuclear cells using the culture system described in the first aspect of the invention. The method according to embodiments of the invention can improve the efficiency of reprogramming peripheral blood mononuclear cells into induced pluripotent stem cells.

[0010] In a fourth aspect, the present invention provides a method for detecting the reprogramming efficiency of peripheral blood mononuclear cells. According to an embodiment of the invention, the method includes reprogramming peripheral blood mononuclear cells using the method described in the third aspect of the invention; and detecting pluripotency markers in the reprogrammed cells. The method according to an embodiment of the invention is capable of detecting the reprogramming efficiency of peripheral blood mononuclear cells.

[0011] In a fifth aspect, the present invention provides an induced pluripotent stem cell. According to an embodiment of the present invention, the induced pluripotent stem cells are obtained by the method described in the third aspect of the present invention. The induced pluripotent stem cells according to the embodiments of the present invention are abundant, highly pure, and free of contaminating cells, thus meeting clinical needs.

[0012] In a sixth aspect, the present invention proposes the application of the induced pluripotent stem cells described in the fifth aspect of the present invention in regenerative medicine.

[0013] Additional aspects and advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention. Attached Figure Description

[0014] The above and / or additional aspects and advantages of the present invention will become apparent and readily understood from the description of the embodiments taken in conjunction with the following drawings, in which:

[0015] Figure 1 This is a timeline diagram illustrating the reprogramming of PBMCs into iPSCs in Example 1 and Comparative Examples 1-3.

[0016] Figure 2 This is a comparison of the effect of PBMC culture medium used in Example 1 and Comparative Example 2 on the proliferation of CD34-positive cells in PBMC.

[0017] Figure 3This is a schematic diagram showing the morphology of iPSCs at different stages in Example 1 and Comparative Examples 1-3, observed using a Leica inverted phase contrast microscope (DMIL-LED).

[0018] Figure 4 The image shows the results of preliminary identification of the iPSCs prepared in Example 1 using an alkaline phosphatase staining kit (beyotime).

[0019] Figure 5 This is a graph showing the results of flow cytometry verification of the positive rates of cell surface markers and internal markers of iPSCs in Example 1.

[0020] Figure 6 This is a graph showing the results of verifying the positive rate of pluripotent markers for iPSCs using immunofluorescence staining in Example 1.

[0021] Figure 7 In Example 1, PCR was used to detect the expression of pluripotent genes in iPSCs.

[0022] Figure 8 The iPSCs were identified using karyotype testing in Example 1. Detailed Implementation

[0023] The embodiments of the present invention are described in detail below. The embodiments described below are exemplary and are only used to explain the present invention, and should not be construed as limiting the present invention.

[0024] It should be noted that the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of indicated technical features. Therefore, a feature defined as "first" or "second" may explicitly or implicitly include one or more of that feature. Furthermore, in the description of this invention, unless otherwise stated, "a plurality of" means two or more.

[0025] To facilitate understanding of this invention, certain technical and scientific terms are specifically defined below. Unless otherwise expressly defined elsewhere in this invention, all other technical and scientific terms used herein have the meanings commonly understood by one of ordinary skill in the art to which this invention pertains.

[0026] In this invention, the terms "comprising" or "including" are open-ended expressions, meaning they include the contents specified in this invention but do not exclude other aspects.

[0027] In this invention, the terms “optionally,” “optionally,” or “optionally” generally refer to events or conditions described subsequently that may but may not occur, and the description includes both cases in which such events or conditions occur and cases in which such events or conditions do not occur.

[0028] In this invention, the term "peripheral blood mononuclear cell (PBMC)" refers to mononuclear cells isolated from peripheral blood, mainly including lymphocytes, but also including a small number of monocytes, plasma cells, hematopoietic stem cells, and other progenitor cells. The PBMCs used in this invention can be isolated from blood or cryopreserved PBMCs. PBMCs isolated from blood are typically obtained from freshly collected blood samples using Ficoll-Paque density gradient centrifugation.

[0029] In this invention, the term "reprogramming" refers to the process of dedifferentiating somatic cells into pluripotent stem cells.

[0030] In this invention, the term "HDAC inhibitor" can also be referred to as histone deacetylase inhibitor (HDACI), which is a reagent capable of inhibiting the activity of histone deacetylase (HDAC).

[0031] In this invention, the term "GSK-3 inhibitor" refers to an inhibitor of glycogen synthase kinase-3.

[0032] In this invention, the term "MEK inhibitor" refers to an inhibitor of mitogen-activated protein kinase (MAPK / ERK kinase).

[0033] This invention proposes a culture composition, a culture system, a method for reprogramming peripheral blood mononuclear cells, a method for detecting the reprogramming efficiency of peripheral blood mononuclear cells, an induced pluripotent stem cell, and the application of induced pluripotent stem cells in regenerative medicine, which will be described in detail below.

[0034] Culture composition

[0035] In a first aspect, the present invention provides a culture composition. According to embodiments of the invention, the culture composition comprises: a small molecule drug and cytokines, wherein the small molecule drug comprises at least one of LBH589, LY2090314, and Selumetinib; and the cytokines comprise at least one of IL-6, IL-3, IL-15, IL-2, SCF, and Flt3L. The culture composition according to embodiments of the invention optimizes the culture medium of conventional methods, adds various cytokines capable of activating different subsets of PBMCs, shortens the PBMC culture time from seven days to one day, reduces time and financial costs, and avoids the cell sorting step; the addition of the small molecule drug improves the efficiency of reprogramming peripheral blood mononuclear cells into induced pluripotent stem cells.

[0036] According to an embodiment of the present invention, the culture composition further comprises small molecule additives.

[0037] According to an embodiment of the present invention, the small molecule additive comprises at least one of 740Y-P and butyramide.

[0038] According to embodiments of the present invention, the cytokines include IL-6, IL-3, IL-15, IL-2, SCF, and Flt3L, and the small molecule drugs include, but are not limited to, at least one of LBH589, VPA (valproic acid), NaBu (sodium butyrate), TSA (trichostatin A), and SAHA (suberoylanilide hydroxamic acid). The culture composition according to embodiments of the present invention optimizes the culture medium of conventional methods, adds various cytokines, activates the proliferation of different subpopulations in PBMCs, shortens the PBMC culture time from seven days to one day, reduces time and money costs, and avoids the cell sorting step; it adds small molecule drugs LBH589, VPA (valproic acid), NaBu (sodium butyrate), TSA (trichostatin A), and SAHA (suberoylanilide hydroxamic acid) to improve the efficiency of peripheral blood mononuclear cells reprogramming into induced pluripotent stem cells. Among them, LBH589, VPA (valproic acid), NaBu (sodium butyrate), TSA (trichostatin A), and SAHA (suberoylanilide hydroxamic acid) are HDAC inhibitors that can promote histone acetylation and further improve the efficiency of peripheral blood mononuclear cells reprogramming into induced pluripotent stem cells.

[0039] According to embodiments of the present invention, the cytokines include IL-6, IL-3, IL-15, IL-2, SCF, and Flt3L, and the small molecule drug includes LBH589. The culture composition according to embodiments of the present invention optimizes the culture medium of conventional methods, adds various cytokines, activates the proliferation of different subsets in PBMCs, shortens the PBMC culture time from seven days to one day, reduces time and cost, and avoids the cell sorting step; the addition of the small molecule drug LBH589 improves the efficiency of peripheral blood mononuclear cell reprogramming into induced pluripotent stem cells. LBH589 is an HDAC inhibitor that can promote histone acetylation, further improving the efficiency of peripheral blood mononuclear cell reprogramming into induced pluripotent stem cells.

[0040] According to embodiments of the present invention, the cytokines include IL-6, IL-3, IL-15, IL-2, SCF, and Flt3L, and the small molecule drug includes at least one of LY2090314, CHIR99021, SB216763, TWS119, Tideglusib, and the small molecule compound BIO (6-bromoindirubin-3-oxime). The culture composition according to embodiments of the present invention optimizes the culture medium of conventional methods, adds various cytokines, activates the proliferation of different subpopulations in PBMCs, shortens the PBMC culture time from seven days to one day, reduces time and money costs, and avoids the cell sorting step; it adds at least one of the small molecule drugs LY2090314, CHIR99021, SB216763, TWS119, Tideglusib and the small molecule compound BIO (6-bromoindirubin-3-oxime), thereby improving the efficiency of peripheral blood mononuclear cell reprogramming into induced pluripotent stem cells.

[0041] According to embodiments of the present invention, the cytokines include IL-6, IL-3, IL-15, IL-2, SCF, and Flt3L, and the small molecule drug includes LY2090314. The culture composition according to embodiments of the present invention optimizes the culture medium of conventional methods, adds various cytokines, activates the proliferation of different subsets in PBMCs, shortens the PBMC culture time from seven days to one day, reduces time and cost, and avoids the cell sorting step; the addition of the small molecule drug LY2090314 improves the efficiency of peripheral blood mononuclear cell reprogramming into induced pluripotent stem cells. LY2090314 is a GSK-3 inhibitor that can improve the efficiency of cell conversion into iPSCs and maintain the stemness of induced pluripotent stem cells.

[0042] According to embodiments of the present invention, the cytokines include IL-6, IL-3, IL-15, IL-2, SCF, and Flt3L, and the small molecule drug includes at least one of Selumetinib and PD0325901. The culture composition according to embodiments of the present invention optimizes the culture medium of conventional methods, adds various cytokines, activates the proliferation of different subsets in PBMCs, shortens the PBMC culture time from seven days to one day, reduces time and cost, and avoids the cell sorting step; it adds the small molecule drugs Selumetinib and PD0325901, which can reduce cell dependence on external growth factors and reduce cell proliferation signals, thereby improving the efficiency of peripheral blood mononuclear cell reprogramming into induced pluripotent stem cells.

[0043] According to embodiments of the present invention, the cytokines include IL-6, IL-3, IL-15, IL-2, SCF, and Flt3L, and the small molecule drug includes Selumetinib. The culture composition according to embodiments of the present invention optimizes the culture medium of conventional methods, adds various cytokines, activates the proliferation of different subsets in PBMCs, shortens the PBMC culture time from seven days to one day, reduces time and cost, and avoids the cell sorting step; it adds Selumetinib, a small molecule drug that can reduce cell dependence on external growth factors and reduce cell proliferation signals, thereby improving the efficiency of peripheral blood mononuclear cell reprogramming into induced pluripotent stem cells. Selumetinib is a MEK inhibitor drug that reduces cell dependence on external growth factors and reduces cell proliferation signals, which helps to promote the transition of cell state to pluripotency.

[0044] According to embodiments of the present invention, the cytokines include IL-6, IL-3, IL-15, IL-2, SCF, and Flt3L, and the small molecule drugs include LBH589, LY2090314, and Selumetinib. The culture composition according to embodiments of the present invention optimizes the culture medium of conventional methods, adds various cytokines, activates the proliferation of different subsets in PBMCs, shortens the PBMC culture time from seven days to one day, reduces time and cost, and avoids the cell sorting step; the addition of the small molecule drug LBH589, which can promote histone acetylation, and the GSK-3 inhibitor LY2090314, as well as the small molecule drug Selumetinib, which can reduce cell dependence on external growth factors and reduce cell proliferation signals, further improves the efficiency of peripheral blood mononuclear cell reprogramming into induced pluripotent stem cells.

[0045] According to an embodiment of the present invention, the molar ratio of IL-6, IL-3, IL-15, IL-2, SCF, Flt3L to LBH589 is (18-22 ng): (18-22 ng): (5-15 ng): (5-10 ng): (90-110 ng): (90-110 ng): (2 × 10⁻⁶ ng) 3 ~1×10 4The culture composition according to embodiments of the present invention achieves synergistic effects among the components by precisely proportioning the cytokines IL-6, IL-3, IL-15, IL-2, SCF (stem cell factor), and Flt3L (FMS-like tyrosine kinase receptor-3 ligand) with the small molecule drug LBH589. This synergistic effect is based on the unique functions and complementarity of each component in the cell reprogramming process, jointly promoting the efficient conversion of peripheral blood mononuclear cells (PBMCs) into induced pluripotent stem cells (iPSCs). Specifically, IL-6, IL-3, IL-15, IL-2, SCF, and Flt3L, as cytokines, each play a role in cell growth, survival, proliferation, and differentiation. The combined use of these components can mimic the complex signals received by cells in the in vivo microenvironment, thereby promoting the reprogramming of PBMCs. LBH589, as a small-molecule HDAC inhibitor, enhances the transcriptional activity of reprogramming factors by promoting histone acetylation, altering chromatin structure, and thus improving reprogramming efficiency. In this embodiment of the invention, the mass ratio of cytokines to LBH589 is carefully designed and optimized to ensure that each component can exert the maximum synergistic effect at appropriate concentrations, further improving reprogramming efficiency. Through this synergistic effect, the culture composition of the present invention not only significantly improves the reprogramming efficiency of PBMCs to iPSCs, but also may reduce the time required in the reprogramming process, lower costs, and improve the ease of operation.

[0046] According to an embodiment of the present invention, the concentration ratio of IL-6, IL-3, IL-15, IL-2, SCF, Flt3L and LBH589 is (18-22 ng / mL): (18-22 ng / mL): (5-15 ng / mL): (5-10 ng / mL): (90-110 ng / mL): (90-110 ng / mL): (2-10 nM).

[0047] According to an embodiment of the present invention, the molar ratio of IL-6, IL-3, IL-15, IL-2, SCF, Flt3L and LY2090314 is (18-22 ng): (18-22 ng): (5-15 ng): (5-10 ng): (90-110 ng): (90-110 ng): (5 × 10⁻⁶ ng) 2 ~2×10 3The culture composition according to embodiments of the present invention achieves synergistic effects among the components by precisely proportioning the cytokines IL-6, IL-3, IL-15, IL-2, SCF (stem cell factor), and Flt3L (FMS-like tyrosine kinase receptor-3 ligand) with the small molecule drug LY2090314. This synergistic effect is based on the unique functions and complementarity of each component in the cell reprogramming process, jointly promoting the efficient conversion of peripheral blood mononuclear cells (PBMCs) into induced pluripotent stem cells (iPSCs). Specifically, IL-6, IL-3, IL-15, IL-2, SCF, and Flt3L, as cytokines, each play a role in cell growth, survival, proliferation, and differentiation. Their combined use can mimic the complex signals received by cells in the in vivo microenvironment, thereby promoting the reprogramming of PBMCs; LY2090314, as a small molecule GSK-3 inhibitor, can improve the efficiency of cell conversion into iPSCs and maintain the stemness of induced pluripotent stem cells; in the embodiments of the present invention, the mass ratio of cytokines to LY2090314 is carefully designed and optimized to ensure that each component can exert the maximum synergistic effect at an appropriate concentration, further improving the reprogramming efficiency; through this synergistic effect, the culture composition of the present invention not only significantly improves the reprogramming efficiency of PBMCs to iPSCs, but also may reduce the time required for the reprogramming process, reduce costs, and improve the ease of operation.

[0048] According to an embodiment of the present invention, the concentration ratio of IL-6, IL-3, IL-15, IL-2, SCF, Flt3L and LY2090314 is (18-22 ng / mL): (18-22 ng / mL): (5-15 ng / mL): (5-10 ng / mL): (90-110 ng / mL): (90-110 ng / mL): (0.5-2 μM).

[0049] According to an embodiment of the present invention, the molar ratio of IL-6, IL-3, IL-15, IL-2, SCF, Flt3L to Selumetinib is (18-22 ng): (18-22 ng): (5-15 ng): (5-10 ng): (90-110 ng): (90-110 ng): (5 × 10⁻⁶ ng) 3 ~1.5×10 4The culture composition according to embodiments of the present invention achieves synergistic effects among the components by precisely proportioning the cytokines IL-6, IL-3, IL-15, IL-2, SCF (stem cell factor), and Flt3L (FMS-like tyrosine kinase receptor-3 ligand) with the small molecule drug Selumetinib. This synergistic effect is based on the unique functions and complementarity of each component in the cell reprogramming process, jointly promoting the efficient conversion of peripheral blood mononuclear cells (PBMCs) into induced pluripotent stem cells (iPSCs). Specifically, IL-6, IL-3, IL-15, IL-2, SCF, and Flt3L, as cytokines, each play a role in cell growth, survival, proliferation, and differentiation. Their combined use can mimic the complex signals received by cells in the in vivo microenvironment, thereby promoting the reprogramming of PBMCs. Selumetinib, as a MEK inhibitor, reduces the cell's dependence on external growth factors and lowers cell proliferation signals, which helps to promote the transition of cell state to pluripotency. In the embodiments of the present invention, the mass ratio of cytokines to Selumetinib is carefully designed and optimized to ensure that each component can exert the maximum synergistic effect at an appropriate concentration, further improving the reprogramming efficiency. Through this synergistic effect, the culture composition of the present invention not only significantly improves the reprogramming efficiency of PBMCs to iPSCs, but also may reduce the time required for the reprogramming process, reduce costs, and improve the ease of operation.

[0050] According to an embodiment of the present invention, the concentration ratio of IL-6, IL-3, IL-15, IL-2, SCF, Flt3L and Selumetinib is (18-22 ng / mL): (18-22 ng / mL): (5-15 ng / mL): (5-10 ng / mL): (90-110 ng / mL): (90-110 ng / mL): (5-15 μM).

[0051] According to an embodiment of the present invention, the molar ratio of IL-6, IL-3, IL-15, IL-2, SCF, Flt3L, LBH589, LY2090314, and Selumetinib is (18-22 ng): (18-22 ng): (5-15 ng): (5-10 ng): (90-110 ng): (90-110 ng): (2 × 10⁻⁶ ng) 3 ~1×10 4 nmol): (5×10 2 ~2×10 3 μmol): (5×10 3 ~1.5×10 4The culture composition according to embodiments of the present invention achieves synergistic effects among the components by precisely proportioning the cytokines IL-6, IL-3, IL-15, IL-2, SCF (stem cell factor), and Flt3L (FMS-like tyrosine kinase receptor-3 ligand) with the small molecule drugs LBH589, LY2090314, and Selumetinib. This synergistic effect is based on the unique function and complementarity of each component in the cell reprogramming process, jointly promoting the efficient conversion of peripheral blood mononuclear cells (PBMCs) into induced pluripotent stem cells (iPSCs).

[0052] According to an embodiment of the present invention, the concentration ratio of IL-6, IL-3, IL-15, IL-2, SCF, Flt3L, LBH589, LY2090314 and Selumetinib is (18-22 ng / mL): (18-22 ng / mL): (5-15 ng / mL): (5-10 ng / mL): (90-110 ng / mL): (90-110 ng / mL): (2-10 nM): (0.5-2 μM): (5-15 μM).

[0053] According to an embodiment of the present invention, the mass molar ratio of IL-6, IL-3, IL-15, IL-2, SCF, Flt3L, LBH589, LY2090314, Selumetinib, 740Y-P, and butyramide is (18-22 ng): (18-22 ng): (5-15 ng): (5-10 ng): (90-110 ng): (90-110 ng): (2 × 10⁻⁶ ng) 3 ~1×10 4 nmol): (5×10 2 ~2×10 3 μmol): (5×10 3 ~1.5×10 4 μmol): (5×10 2 ~2×10 3(50-200 μmol). The culture composition according to embodiments of the present invention achieves synergistic effects among these components by precisely proportioning cytokines IL-6, IL-3, IL-15, IL-2, SCF (stem cell factor), and Flt3L (FMS-like tyrosine kinase receptor-3 ligand) with small molecule drugs LBH589, LY2090314, Selumetinib, and small molecule additives 740Y-P and butyramide. This synergistic effect is based on the unique function and complementarity of each component in the cell reprogramming process, jointly promoting the efficient conversion of peripheral blood mononuclear cells (PBMCs) into induced pluripotent stem cells (iPSCs).

[0054] According to an embodiment of the present invention, the concentration ratio of IL-6, IL-3, IL-15, IL-2, SCF, Flt3L, LBH589, LY2090314, Selumetinib, 740Y-P and butyramide is (18-22 ng / mL): (18-22 ng / mL): (5-15 ng / mL): (5-10 ng / mL): (90-110 ng / mL): (90-110 ng / mL): (2-10 nM): (0.5-2 μM): (5-15 μM): (0.5-2 μM): (0.05-0.2 μM).

[0055] training system

[0056] In a second aspect, the present invention provides a culture system. According to embodiments of the present invention, the culture system comprises a culture medium and the culture composition described in the first aspect of the present invention. The culture system according to embodiments of the present invention optimizes the culture medium of conventional methods, adds various cytokines capable of activating different subsets of PBMCs, shortens the PBMC culture time from seven days to one day, reduces time and financial costs, and avoids the cell sorting step; it also adds small molecule drugs that can improve the efficiency of reprogramming peripheral blood mononuclear cells into induced pluripotent stem cells.

[0057] According to an embodiment of the present invention, the culture system further includes a transducer virus.

[0058] According to an embodiment of the present invention, the transduction virus is selected from at least one of Sendai virus, lentivirus, retrovirus, adenovirus, and adeno-associated virus.

[0059] According to embodiments of the present invention, the culture medium includes RPMI 1640 medium, DMEM medium, F12 medium, and StemSpan medium. TMAt least one of the following: SFEMII medium. It should be noted that RPMI 1640 medium was originally used for the growth and serum-free culture expansion of human lymphocytes. With appropriate nutrient supplementation, RPMI 1640 medium can be widely used for the culture of various mammalian cells.

[0060] According to embodiments of the present invention, the concentration of IL-6 in the culture system is 15–25 ng / mL, for example, 15 ng / mL, 16 ng / mL, 17 ng / mL, 18 ng / mL, 19 ng / mL, 20 ng / mL, 21 ng / mL, 22 ng / mL, 23 ng / mL, 24 ng / mL, 25 ng / mL, or a range between the two, such as 16–25 ng / mL, 17–25 ng / mL, and 18–25 ng / mL. Therefore, an appropriate concentration of IL-6 can activate the proliferation of different subpopulations in PBMCs, shorten the PBMC culture time, reduce time and cost, and avoid the cell sorting step.

[0061] According to embodiments of the present invention, the concentration of IL-3 in the culture system is 15–25 ng / mL, for example, 15 ng / mL, 16 ng / mL, 17 ng / mL, 18 ng / mL, 19 ng / mL, 20 ng / mL, 21 ng / mL, 22 ng / mL, 23 ng / mL, 24 ng / mL, 25 ng / mL, or a range between the two, such as 16–25 ng / mL, 17–25 ng / mL, and 18–25 ng / mL. Therefore, an appropriate concentration of IL-3 can activate the proliferation of different subpopulations in PBMCs, shorten the PBMC culture time, reduce time and cost, and avoid the cell sorting step.

[0062] According to embodiments of the present invention, the concentration of IL-15 in the culture system is 5–15 ng / mL, for example, 5 ng / mL, 6 ng / mL, 7 ng / mL, 8 ng / mL, 9 ng / mL, 10 ng / mL, 11 ng / mL, 12 ng / mL, 13 ng / mL, 14 ng / mL, 15 ng / mL, or a range between the two, such as 6–15 ng / mL, 7–15 ng / mL, and 8–15 ng / mL. Thus, an appropriate concentration of IL-15 can activate the proliferation of different subpopulations in PBMCs, shorten the PBMC culture time, reduce time and cost, and avoid the cell sorting step.

[0063] According to an embodiment of the present invention, the concentration of IL-2 in the culture system is 5–10 ng / mL, for example, 5 ng / mL, 6 ng / mL, 7 ng / mL, 8 ng / mL, 9 ng / mL, 10 ng / mL, or a range between the two, 6–10 ng / mL. Therefore, an appropriate concentration of IL-2 can activate the proliferation of different subpopulations in PBMCs, shorten the PBMC culture time, reduce time and cost, and avoid cell sorting steps.

[0064] According to embodiments of the present invention, the concentration of SCF in the culture system is 90–110 ng / mL, for example, it can be 90 ng / mL, 91 ng / mL, 92 ng / mL, 94 ng / mL, 96 ng / mL, 98 ng / mL, 100 ng / mL, 102 ng / mL, 104 ng / mL, 106 ng / mL, 108 ng / mL, 109 ng / mL, 110 ng / mL, or a range between the two, such as 91–110 ng / mL and 92–110 ng / mL. Therefore, an appropriate concentration of SCF can activate the proliferation of different subpopulations in PBMCs, shorten the PBMC culture time, reduce time and cost, and avoid cell sorting steps.

[0065] According to embodiments of the present invention, the concentration of Flt3L in the culture system is 90–110 ng / mL, for example, it can be 90 ng / mL, 91 ng / mL, 92 ng / mL, 94 ng / mL, 96 ng / mL, 98 ng / mL, 100 ng / mL, 102 ng / mL, 104 ng / mL, 106 ng / mL, 108 ng / mL, 109 ng / mL, 110 ng / mL, or a range between the two, such as 91–110 ng / mL and 92–110 ng / mL. Therefore, an appropriate concentration of Flt3L can activate the proliferation of different subpopulations in PBMCs, shorten the PBMC culture time, reduce time and cost, and avoid cell sorting steps.

[0066] According to embodiments of the present invention, the concentration of 740Y-P in the culture system is 0.5–2 μM, for example, it can be 0.5 μM, 0.6 μM, 0.7 μM, 0.8 μM, 0.9 μM, 1.0 μM, 1.1 μM, 1.2 μM, 1.3 μM, 1.4 μM, 1.5 μM, 1.6 μM, 1.7 μM, 1.8 μM, 1.9 μM, 2.0 μM, or a range between the two, 0.6–2 μM and 0.7–2 μM. Therefore, a suitable concentration of 740Y-P can activate the proliferation of different subpopulations in PBMCs, shorten the PBMC culture time, reduce time and money costs, and avoid cell sorting steps.

[0067] According to embodiments of the present invention, the concentration of butyramide in the culture system is 0.05–0.2 μM, for example, 0.05 μM, 0.06 μM, 0.07 μM, 0.08 μM, 0.09 μM, 0.1 μM, 0.2 μM, or a range between the two, 0.06–0.2 μM and 0.07–0.2 μM. Thus, butyramide at an appropriate concentration can activate the proliferation of different subpopulations in PBMCs, shorten the PBMC culture time, reduce time and cost, and avoid cell sorting steps.

[0068] According to an embodiment of the present invention, the concentration of LBH589 in the culture system is 1–10 nM, for example, it can be 1 nM, 2 nM, 3 nM, 4 nM, 5 nM, 6 nM, 7 nM, 8 nM, 9 nM, 10 nM, or a range between the two, such as 2–10 nM and 3–10 nM. Thus, an appropriate concentration of LBH589 can promote histone acetylation, further improving the efficiency of peripheral blood mononuclear cell reprogramming into induced pluripotent stem cells.

[0069] According to embodiments of the present invention, the concentration of LY2090314 in the culture system is 0.01–5 μM, for example, it can be 0.01 μM, 0.02 μM, 0.03 μM, 0.04 μM, 0.05 μM, 0.06 μM, 0.07 μM, 0.08 μM, 0.09 μM, 0.1 μM, 0.2 μM, 0.3 μM, 0.4 μM, 0.5 μM, 0.6 μM, 0.7 μM, 0.8 μM, 0.9 μM, 1 μM, 2 μM, 3 μM, 4 μM, 5 μM, or a range between 0.02–5 μM and 0.03–5 μM. Therefore, an appropriate concentration of LY2090314 can improve the efficiency of cell conversion into iPSCs and maintain the stemness of induced pluripotent stem cells.

[0070] According to embodiments of the present invention, the concentration of Selumetinib in the culture system is 1–20 μM, for example, it can be 1 μM, 2 μM, 3 μM, 4 μM, 5 μM, 6 μM, 7 μM, 8 μM, 9 μM, 10 μM, 11 μM, 12 μM, 13 μM, 14 μM, 15 μM, 16 μM, 17 μM, 18 μM, 19 μM, 20 μM, or a range between the two, 2–20 μM and 3–20 μM. Thus, an appropriate concentration of Selumetinib can reduce the cell's dependence on external growth factors and decrease cell proliferation signals, which helps to promote the transition of the cell state to a pluripotent state.

[0071] Methods for reprogramming peripheral blood mononuclear cells

[0072] In a third aspect, the present invention provides a method for reprogramming peripheral blood mononuclear cells. According to embodiments of the invention, the method includes culturing peripheral blood mononuclear cells using the culture system described in the first aspect of the invention. The method according to embodiments of the invention can improve the efficiency of reprogramming peripheral blood mononuclear cells into induced pluripotent stem cells.

[0073] According to an embodiment of the present invention, the culture treatment is carried out by pre-culturing peripheral blood mononuclear cells in a culture medium comprising the cytokines and small molecule additives; and transducing the pre-cultured peripheral blood mononuclear cells using Sendai virus and a small molecule drug.

[0074] According to an embodiment of the present invention, the peripheral blood mononuclear cells are obtained by gradient centrifugation.

[0075] According to an embodiment of the present invention, the culture treatment time is 20 to 28 hours.

[0076] According to embodiments of the present invention, the cytokines include at least one of IL-6, IL-3, IL-15, IL-2, SCF, and Flt3L.

[0077] According to embodiments of the present invention, the cytokines include IL-6, IL-3, IL-15, IL-2, SCF, and Flt3L, and the small molecule drug includes LBH589.

[0078] According to embodiments of the present invention, the cytokines include IL-6, IL-3, IL-15, IL-2, SCF, and Flt3L, and the small molecule drug includes LY2090314.

[0079] According to embodiments of the present invention, the cytokines include IL-6, IL-3, IL-15, IL-2, SCF, and Flt3L, and the small molecule drug includes Selumetinib.

[0080] According to an embodiment of the present invention, the molar ratio of IL-6, IL-3, IL-15, IL-2, SCF, Flt3L to LBH589 is (18-22 ng): (18-22 ng): (5-15 ng): (5-10 ng): (90-110 ng): (90-110 ng): (2 × 10⁻⁶ ng) 3 ~1×10 4 nmol).

[0081] According to an embodiment of the present invention, the concentration ratio of IL-6, IL-3, IL-15, IL-2, SCF, Flt3L and LBH589 is (18-22 ng / mL): (18-22 ng / mL): (5-15 ng / mL): (5-10 ng / mL): (90-110 ng / mL): (90-110 ng / mL): (2-10 nM).

[0082] According to an embodiment of the present invention, the molar ratio of IL-6, IL-3, IL-15, IL-2, SCF, Flt3L and LY2090314 is (18-22 ng): (18-22 ng): (5-15 ng): (5-10 ng): (90-110 ng): (90-110 ng): (5 × 10⁻⁶ ng) 2 ~2×10 3 μmol).

[0083] According to an embodiment of the present invention, the concentration ratio of IL-6, IL-3, IL-15, IL-2, SCF, Flt3L and LY2090314 is (18-22 ng / mL): (18-22 ng / mL): (5-15 ng / mL): (5-10 ng / mL): (90-110 ng / mL): (90-110 ng / mL): (0.5-2 μM).

[0084] According to an embodiment of the present invention, the molar ratio of IL-6, IL-3, IL-15, IL-2, SCF, Flt3L to Selumetinib is (18-22 ng): (18-22 ng): (5-15 ng): (5-10 ng): (90-110 ng): (90-110 ng): (5 × 10⁻⁶ ng) 3 ~1.5×10 4 μmol).

[0085] According to an embodiment of the present invention, the concentration ratio of IL-6, IL-3, IL-15, IL-2, SCF, Flt3L and Selumetinib is (18-22 ng / mL): (18-22 ng / mL): (5-15 ng / mL): (5-10 ng / mL): (90-110 ng / mL): (90-110 ng / mL): (5-15 μM).

[0086] According to an embodiment of the present invention, the molar ratio of IL-6, IL-3, IL-15, IL-2, SCF, Flt3L, LBH589, LY2090314, and Selumetinib is (18-22 ng): (18-22 ng): (5-15 ng): (5-10 ng): (90-110 ng): (90-110 ng): (2 × 10⁻⁶ ng) 3 ~1×10 4 nmol): (5×10 2 ~2×10 3 μmol): (5×10 3 ~1.5×10 4 μmol).

[0087] According to an embodiment of the present invention, the concentration ratio of IL-6, IL-3, IL-15, IL-2, SCF, Flt3L, LBH589, LY2090314 and Selumetinib is (18-22 ng / mL): (18-22 ng / mL): (5-15 ng / mL): (5-10 ng / mL): (90-110 ng / mL): (90-110 ng / mL): (2-10 nM): (0.5-2 μM): (5-15 μM).

[0088] According to an embodiment of the present invention, the mass molar ratio of IL-6, IL-3, IL-15, IL-2, SCF, Flt3L, LBH589, LY2090314, Selumetinib, 740Y-P, and butyramide is (18-22 ng): (18-22 ng): (5-15 ng): (5-10 ng): (90-110 ng): (90-110 ng): (2 × 10⁻⁶ ng) 3 ~1×10 4 nmol): (5×10 2 ~2×10 3 μmol): (5×10 3 ~1.5×10 4 μmol): (5×10 2 ~2×10 3 μmol); (50~200μmol).

[0089] According to an embodiment of the present invention, the concentration ratio of IL-6, IL-3, IL-15, IL-2, SCF, Flt3L, LBH589, LY2090314, Selumetinib, 740Y-P and butyramide is (18-22 ng / mL): (18-22 ng / mL): (5-15 ng / mL): (5-10 ng / mL): (90-110 ng / mL): (90-110 ng / mL): (2-10 nM): (0.5-2 μM): (5-15 μM): (0.5-2 μM): (0.05-0.2 μM).

[0090] According to an embodiment of the present invention, the concentration of IL-6 in the culture medium is 15-25 ng / mL.

[0091] According to an embodiment of the present invention, the concentration of IL-3 in the culture medium is 15-25 ng / mL.

[0092] According to an embodiment of the present invention, the concentration of IL-15 in the culture medium is 5 to 15 ng / mL.

[0093] According to an embodiment of the present invention, the concentration of IL-2 in the culture medium is 5 to 10 ng / mL.

[0094] According to an embodiment of the present invention, the concentration of SCF in the culture medium is 90-110 ng / mL.

[0095] According to an embodiment of the present invention, the concentration of Flt3L in the culture medium is 90-110 ng / mL.

[0096] According to an embodiment of the present invention, the concentration of 740Y-P in the culture medium is 0.5 to 2 μM.

[0097] According to an embodiment of the present invention, the concentration of butyramide in the culture medium is 0.05 to 0.2 μM.

[0098] According to an embodiment of the present invention, the concentration of LBH589 in the culture medium is 1 to 10 nM.

[0099] According to an embodiment of the present invention, the concentration of LY2090314 in the culture medium is 0.01 to 5 μM.

[0100] According to an embodiment of the present invention, the concentration of Selumetinib in the culture medium is 1–20 μM.

[0101] Methods for detecting the reprogramming efficiency of peripheral blood mononuclear cells

[0102] In a fourth aspect, the present invention provides a method for detecting the reprogramming efficiency of peripheral blood mononuclear cells. According to an embodiment of the invention, the method includes reprogramming peripheral blood mononuclear cells using the method described in the third aspect of the invention; and detecting pluripotency markers in the reprogrammed cells. The method according to an embodiment of the invention is capable of detecting the reprogramming efficiency of peripheral blood mononuclear cells.

[0103] Induced pluripotent stem cells

[0104] In a fifth aspect, the present invention provides an induced pluripotent stem cell. According to an embodiment of the present invention, the induced pluripotent stem cells are obtained by the method described in the third aspect of the present invention. The induced pluripotent stem cells according to the embodiments of the present invention are abundant, highly pure, and free of contaminating cells, thus meeting clinical needs.

[0105] Induced pluripotent stem cells and their application in regenerative medicine

[0106] In a sixth aspect, the present invention proposes the application of the induced pluripotent stem cells described in the fifth aspect of the present invention in regenerative medicine.

[0107] The present invention will be explained below with reference to embodiments. Those skilled in the art will understand that the following embodiments are for illustrative purposes only and should not be considered as limiting the scope of the invention. Where specific techniques or conditions are not specified in the embodiments, they are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be obtained commercially.

[0108] Example 1:

[0109] 1. Peripheral blood mononuclear cells (PBMCs) were isolated from peripheral blood using gradient centrifugation.

[0110] 1.1 Add Ficoll separation solution to a 15 mL centrifuge tube (the amount should be approximately the same as the volume of peripheral blood to be extracted). Tilt the centrifuge tube and slowly add the peripheral blood along the side wall. Centrifuge at 2500 rpm for 25 minutes at room temperature.

[0111] 1.2 Gently transfer the centrifuge tube to the biosafety cabinet. Use a 1mL pipette tip to gently aspirate the top layer of liquid, being careful not to aspirate the white membrane layer. Then, use a yellow pipette tip to gently aspirate the cells from the white membrane layer. The aspirated cells are PBMCs.

[0112] 2. PBMC pre-culture

[0113] PBMCs were pre-cultured for 24 h in a medium containing the following formulation: basal medium: RPMI 1640 medium (Gibco, 11875101); medium supplements: 20 ng / mL interleukin-6 (IL-6) (Peprotech, 200-06), 20 ng / mL interleukin-3 (IL-3) (Peprotech, 200-03), 5-15 ng / mL interleukin-15 (IL-15) (Peprotech, 200-15), 5-10 ng / mL interleukin-2 (IL-2) (Peprotech, 200-02), 100 ng / mL stem cell factor (SCF) (Peprotech, 300-07), 100 ng / mL FMS-like tyrosine kinase receptor-3 ligand (Flt3L) (Peprotech, 300-19), 1 μM 740Y-P (MedChemExpress, HY-P0175), 0.1 μM butanamide (Aladdin, B152329).

[0114] 3. Sendai virus transduction

[0115] 3.1 AOPI counting was performed on PBMCs that had been pre-cultured for 24 hours.

[0116] 3.2 The volume of virus added was calculated based on MOI = 5:5:3 (KOS:hc-Myc:Klf4), viral titer, and cell count. (Formula and titer are from CyToTune) TM (As provided in the Sendai test tube instructions, version 2.0).

[0117] 3.3 Absorb 5×10 4 A cell suspension of one cell was added to a sterile tube.

[0118] 3.4 Remove CyToTune from -80°C TM 2.0 Sendai test tube. After thawing, briefly centrifuge and immediately place on ice.

[0119] 3.5 Calculate the corresponding Sendai virus (Invitrogen, A16517) volume and add it to 300 μL of basal medium containing 5 nM LBBH589, 1 μM LY2090314, and 10 μM Selumetinib from the pre-cultured PBMCs in step 2. Mix well.

[0120] 3.6 Add the reprogrammed virus mixture (step 3.5) to the cell suspension (step 3.3), mix thoroughly, and transfer to a 24-well plate. Incubate at 37°C in a 5% CO2 incubator for two days.

[0121] 4. Remove viruses

[0122] 4.1 After 48 hours of cell culture, transfer the supernatant from the 24-well plate to the corresponding 15 mL centrifuge tubes. Centrifuge at 400 g for 10 min to remove CytoTune. TM Sendai virus 2.0. Discard the supernatant.

[0123] 4.2 Resuspend the cells in 500 μL of PBMC medium (step 2) containing 5 nM LBH589 (Selleck, S1030), 1 μM LY2090314 (Selleck, S7063), and 10 μM Selumetinib (MedChemExpress, S1008), and add the cells to the corresponding wells. Incubate the cells at 37°C in a 5% CO2 incubator for two days.

[0124] 4.3 Coat 12-well plates with 500 μL of Laminin (Biolamina, LN521-02) at a final concentration of 10 μg / mL and incubate overnight at 4°C.

[0125] 5. Cell seeding

[0126] 5.1 Collect the cell supernatant into a 15 mL centrifuge tube. Wash the cells three times with 500 μL of DPBS in a well plate. Add 250 μL of Accutase (Gibco, A1110501) and digest at room temperature for 1-2 min. Add 500 μL of complete culture medium to stop the digestion and collect the cells into the 15 mL centrifuge tube. Centrifuge at 200 g for 5 min.

[0127] 5.2 Discard the supernatant and resuspend the cells in RPMI 1640 medium containing 10 μM Y-27632, 5 nM LBH589, 1 μM LY2090314, and 10 μM elumetinib. Perform AOPI counts.

[0128] 5.3 For each Laminin-coated 12-well plate, the supernatant was aspirated, and cells were distributed at a ratio of 5 × 10⁶ cells per well. 4 Seeds were seeded into 12-well plates coated with laminin. The plates were shaken evenly in a cross pattern. The plates were then incubated at 37°C in a 5% CO2 incubator.

[0129] 6. Replace half the culture medium.

[0130] One day later, 500 μL of the solution was aspirated from each well of the 12-well plate and placed in a 15 mL centrifuge tube. The plate was centrifuged at 200 g for 10 min. The supernatant was discarded, and the solution was resuspended in 500 μL of fresh RPMI 1640 medium containing 5 nM LBH589, 1 μM LY2090314, and 10 μM Selumetinib. The solution was then added to the corresponding well.

[0131] 7. Transitioning in iPSCs culture medium

[0132] One day later, 500 μL of the culture medium was removed and 500 μL of fresh iPSC medium was added. The mixture was incubated overnight at 37°C with 5% CO2. The iPSC medium formula is as follows: mTeSR TM 1Basal Medium (STEMCELL, 85851), mTeSR TM 1 5X supplement (STEMCELL, 85852), KnockOut Serum Replacement (Gibco, 10828-028), Penicillin-Streptomycin 100X (Gibco, 15140122).

[0133] 8. Culturing and monitoring cells

[0134] One day later, discard the old culture medium and replace it with 1 mL of iPSC medium. Change the culture medium daily. Once cells have formed clones, select clones for passage.

[0135] 9. Selecting clones

[0136] 9.1 Remove the laminin-coated 6-well plate from the 4°C freezer in advance and allow it to warm to room temperature. After removing the supernatant, add 1 mL of iPSC complete culture medium to each well.

[0137] 9.2 Observe the cells under an inverted microscope. Mark the colonies to be selected at the bottom of the culture plate. Gently streak along the marked circle with a 2 mL syringe needle, and make parallel and vertical cuts within the circle. Press the colony down with a 200 μL pipette tip, gently lift it, and transfer it to a 6-well plate. Swish the plate a few times to allow the cells to enter the culture medium. Do not streak into laminin. Resuspend the cells in 1 mL of culture medium and incubate at 37°C in a 5% CO2 incubator.

[0138] Comparative Example 1:

[0139] The experimental procedure for this comparative example is basically the same as that for Example 1, except that the PBMCs were pre-cultured for 5 days in a medium containing the following formulation: basal medium: StemSpan™ SFEMII medium; medium supplement: StemSpan™ CD34. + Expansion Supplement (10X), and during Sendai virus transduction, PBMC cells that had been pre-cultured for 5 days were counted using AOPI, with the other procedures being the same as in Example 1.

[0140] Comparative Example 2:

[0141] The experimental procedure for this comparative example is basically the same as that for Example 1, except that the PBMCs were pre-cultured for 1 day in a medium containing the following formulation: basal medium: StemSpan TM SFEMII medium, medium supplement: StemSpan TM CD34 + Expansion Supplement (10X).

[0142] PBMC pre-culture followed by CD34 + Cell sorting, CD34 + The cell sorting process is shown below:

[0143] (1) Gently tap the cell culture flask. Tap off some adherent cells, collect PBMCs into a 50mL centrifuge tube, centrifuge at 200g for 10 minutes, and discard the supernatant. Use StemSpan... TM Resuspend PBMCs in SFEMII medium into 50 mL centrifuge tubes, count cells using AOPI, centrifuge at 200 g for 10 min, and discard the supernatant. If the initial cell count is <1×10⁻⁶, 7 Resuspend the cells in 0.1 mL RoboSep Buffer; if the initial cell number is 1 × 10⁶ cells / mL. 7 -1×10 8 cells, resuspend cells at 1×10⁻⁶ 8 cells / mL RoboSep Buffer; if the initial cell number is 1-5 × 10⁻⁵ 8 Resuspend the cells in 1 mL RoboSep Buffer and transfer them to a 5 mL (12 x 75 mm) flow cytometer tube.

[0144] (2) Add 100 μL / mL of Selection Cocktail to the sample, mix well and incubate at room temperature for 10 minutes.

[0145] (3) Using RapidSpheres TM First, vortex oscillate for 30 seconds to evenly disperse the magnetic beads. Then add RapidSpheres to the sample. TM 100 μL / mL, mix well and incubate at room temperature for 5 minutes.

[0146] (4) Using RoboSep TM Add the sample mixture to a final volume of 2.5 mL using Buffer (Catalog #20104), and gently mix using a pipette, pipetting 2-3 times up and down. Place the flow cytometer (without cap) into the magnet, inserting the tube all the way in. Incubate at room temperature for 3 minutes.

[0147] (5) Keep the flow cytometer tube inserted in the magnet, tilt the entire magnet and flow cytometer tube, pour out the supernatant, hold the tilting position for 2-3 seconds, and let it flow down naturally. Do not shake or use a pipette to aspirate. Remove the flow cytometer tube from the magnet. The flow cytometer tube contains the sorted target cells.

[0148] (6) Repeat steps (4) and (5) four times, for a total of 5×3min of sorting.

[0149] (7) Resuspend the cells in the basal medium of the pre-cultured PBMCs described above, ensuring that the cells are collected from the tube wall. Obtain the target cell suspension. Perform AOPI counting; other procedures are the same as in Example 1.

[0150] Comparative Example 3:

[0151] The experimental process of this comparative example is basically the same as that of Example 1. The difference is that during the Sendai virus transduction process, 5 nM LBH589, 1 μM LY2090314 and 10 μM Selumetinib are not added to the basal medium of the pre-cultured PBMC in step 2. The other processes are the same as those in Example 1.

[0152] Example 1 ( Figure 1 A) Comparative Example 1 Figure 1 B) Comparative Example 2 Figure 1 C) Comparative Example 3 Figure 1 D) The timeline of the method used to reprogram the PBMC into iPSCs is as follows: Figure 1 As shown, Comparative Example 1 required approximately 11-13 days before being completely replaced with iPSCs culture medium, while the culture method and system used in Example 1 of this invention halved the time required for this process and eliminated the need for additional CD34 sorting. + This invention utilizes cells to improve reprogramming efficiency, demonstrating that it has developed a time-saving, low-cost, simple, and efficient method for reprogramming peripheral blood mononuclear cells into induced pluripotent stem cells.

[0153] The effect of the PBMC culture medium used in Example 1 and Comparative Example 2 on the proliferation of CD34-positive cells in PBMCs was compared using flow cytometry. Figure 2 As shown, the proportion of CD34-positive cells was higher in Example 1. This verifies that the PBMC culture medium and its additives used in this invention significantly increased the proliferation of CD34-positive cells in PBMCs and increased the probability of PBMCs being induced into iPSCs.

[0154] The morphology of iPSCs at different stages was observed using a Leica inverted phase contrast microscope (DMIL-LED): For the iPSCs cultured in Example 1, Figure 3A shows the cell morphology on day four after viral transduction. After being transferred to a Laminin 521-coated plate and cultured for 12 days, numerous adherent and aggregated cells appeared. Figure 3 In sample B, iPSC-like clones have already begun to appear. The morphology of the iPSCs was observed on day 22 after the first selection and passage of clones. Figure 3 In cell C, the cell morphology appears as a dense, cobblestone pattern. After selection and purification of clones during passage, Figure 3 As shown in Figure D, the iPSCs cultured to passage P10 exhibit clear clonal edges, high purity, and absence of contaminating cells. For the iPSCs cultured in Comparative Example 1,... Figure 3 E represents the cell morphology on day four after viral transduction. Subsequent cells were transferred to wells coated with Laminin 521. Figure 3 As seen in Figure F, no cells adhered and grew, indicating cell induction failure and poor reproducibility of this experimental method. For the iPSCs cultured in Comparative Example 2, Figure 3 G shows the cell morphology on the fourth day after viral transduction. Figure 3 As shown in H, the cells were transferred to a well plate coated with Laminin 521 and cultured. Cell morphology... Figure 3 I shows the cell morphology after the first passage and the cells cultured to... Figure 3 The cell clonal morphology of iPSCs after generation P10 shown in J is consistent with that in Example 1. For the iPSCs cultured in Comparative Example 3, Figure 3 K represents the cell morphology on the fourth day after viral transduction, such as... Figure 3 The cells shown in Figure L, after being transferred to well plates coated with Laminin 521 and cultured for 16 days, still failed to produce iPSC-like cells; only a small clump of dead cells was visible in the entire field of view, indicating cell induction failure. These experimental results demonstrate that the method of this invention efficiently generates induced pluripotent stem cells from PBMCs and effectively sorts CD34 cells. + The efficiency of reprogramming cells after reprogramming is comparable.

[0155] Human induced pluripotent stem cells (iPSCs) highly express alkaline phosphatase (AP), just like human embryonic stem cells. Therefore, an alkaline phosphatase staining kit (beyotime) was used to preliminarily identify the iPSCs prepared in Example 1. First, the culture medium for the cells to be stained was aspirated, and the cells were washed twice with PBS. The cells were fixed in 4% paraformaldehyde at room temperature for 20 minutes. The paraformaldehyde fixative was aspirated, and the cells were washed twice with TBST buffer. Then, AP buffer was added, and the cells were equilibrated at room temperature for 5 minutes. During equilibration, the AP chromogenic solution was prepared according to the manufacturer's instructions. The AP buffer was aspirated, and the cells were incubated in the dark for 15 minutes. The AP chromogenic solution was aspirated, and the cells were washed twice with PBS, then PBS was added for further observation. The detection results are as follows: Figure 4 As shown, iPSCs are positive for alkaline phosphatase staining.

[0156] The positivity rate of cell surface markers in the iPSCs cultured in Example 1 was verified by flow cytometry, and the results are as follows: Figure 5 As shown, Example 1 improved the efficiency of induced pluripotent stem cells from PBMCs, resulting in highly pure iPSC cell monoclonals. The positive expression rates of cell surface markers SSEA4 and TRA-1-60 were 99.86% and 90.90%, respectively; the positive expression rates of intracellular markers NANOG and OCT4 were 95.51% and 97.09%, respectively.

[0157] The pluripotency of the iPSCs cultured in Example 1 was verified by immunofluorescence staining, and the results are as follows: Figure 6 As shown, the iPSC clones in Example 1 were positive for immunofluorescence staining for pluripotency markers TRA-1-60, NANOG, SSEA4, and OCT4.

[0158] The expression of pluripotent genes in the iPSCs cultured in Example 1 was detected using PCR. The detection method was as follows: Total RNA was extracted using a kit, and genomic DNA contamination was removed by DNase digestion. Reverse transcription was performed using a reverse transcription kit. Polymerase chain reaction (PCR) was performed using SYBR Green Realtime PCR Master Mix. The PCR conditions were: initial denaturation at 95℃ for 1 min, denaturation at 95℃ for 15 s, annealing at 60℃ for 15 s, extension at 72℃ for 45 s, for 39 cycles. Primer information is shown in Table 1, and the detection results are as follows. Figure 7 As shown, compared to the control group (PBMC), iPSCs expressed the pluripotency markers NANOG and SOX2.

[0159] Table 1: Primer Information

[0160]

[0161]

[0162] The iPSCs cultured using the method in Example 1 were subjected to the following process: Figure 8 The karyotype identification shown did not reveal any abnormalities in chromosome number or structure, confirming that iPSCs cultured using the method in Example 1 can still exhibit a normal karyotype after 10 consecutive generations of culture.

[0163] In summary, all the above results demonstrate that this application successfully and efficiently obtained unintegrated and feeder-free iPSCs from PBMCs using Sendai virus. These cells expressed pluripotency markers at the gene level.

[0164] Compared with Comparative Examples 1 and 2, the method of Example 1 shortens the time required to establish iPSC cell lines from PBMCs, improves induction efficiency, and reduces induction costs. The prepared iPSCs are numerous, highly pure, and free of contaminating cells, meeting clinical needs.

[0165] Compared with Comparative Example 3, Example 1 generated a larger number of clones after viral transduction, and the induction experiment was stable and easily reproducible. This application provides a simple and efficient method for establishing iPSCs from PBMCs, reducing the difficulty of experimental operation and shortening the experimental time. The cell morphology and expression of pluripotency markers of the iPSCs prepared in Example 1 were characterized by a series of methods.

[0166] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "example," "specific example," or "some examples," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of the present invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. Moreover, without contradiction, those skilled in the art can combine and integrate the different embodiments or examples described in this specification, as well as the features of different embodiments or examples.

[0167] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention.

Claims

1. A culture composition, characterized in that, include: The small molecule drug and cytokines, wherein the small molecule drug includes at least one of LBH589, LY2090314 and Selumetinib; and the cytokines include at least one of IL-6, IL-3, IL-15, IL-2, SCF and Flt3L.

2. The culture composition according to claim 1, characterized in that, The culture composition further includes small molecule additives; Optionally, the small molecule additive includes at least one of 740Y-P and butyramide.

3. The culture composition according to claim 2, characterized in that, The cytokines include IL-6, IL-3, IL-15, IL-2, SCF, and Flt3L, and the small molecule drug includes at least one of LBH589, VPA, NaBu, TSA, and SAHA; Optionally, the cytokines include IL-6, IL-3, IL-15, IL-2, SCF, and Flt3L, and the small molecule drug includes at least one of LY2090314, CHIR99021, SB216763, TWS119, Tideglusib, and the small molecule compound BIO. Optionally, the cytokines include IL-6, IL-3, IL-15, IL-2, SCF, and Flt3L, and the small molecule drug includes at least one of Selumetinib and PD0325901; Optionally, the cytokines include IL-6, IL-3, IL-15, IL-2, SCF, and Flt3L, and the small molecule drugs include LBH589, LY2090314, and Selumetinib; Optionally, the molar ratio of IL-6, IL-3, IL-15, IL-2, SCF, Flt3L to LBH589 is (18–22 ng): (18–22 ng): (5–15 ng): (5–10 ng): (90–110 ng): (90–110 ng): (2 × 10⁻⁶ ng) 3 ~1×10 4 nmol); Optionally, the molar ratio of IL-6, IL-3, IL-15, IL-2, SCF, Flt3L to LY2090314 is (18–22 ng): (18–22 ng): (5–15 ng): (5–10 ng): (90–110 ng): (90–110 ng): (5 × 10⁻⁶ ng) 2 ~2×10 3 μmol); Optionally, the molar ratio of IL-6, IL-3, IL-15, IL-2, SCF, Flt3L to Selumetinib is (18–22 ng): (18–22 ng): (5–15 ng): (5–10 ng): (90–110 ng): (90–110 ng): (5 × 10⁻⁶ ng) 3 ~1.5×10 4 μmol); Optionally, the molar ratio of IL-6, IL-3, IL-15, IL-2, SCF, Flt3L, LBH589, LY2090314, and Selumetinib is (18–22 ng): (18–22 ng): (5–15 ng): (5–10 ng): (90–110 ng): (90–110 ng): (2 × 10⁻⁶ ng) 3 ~1×10 4 nmol): (5×10 2 ~2×10 3 μmol): (5×10 3 ~1.5×10 4 μmol); Optionally, the molar ratio of the IL-6, IL-3, IL-15, IL-2, SCF, Flt3L, LBH589, LY2090314, Selumetinib, 740Y-P, and butyramide is (18–22 ng): (18–22 ng): (5–15 ng): (5–10 ng): (90–110 ng): (90–110 ng): (2 × 10⁻⁶ ng) 3 ~1×10 4 nmol): (5×10 2 ~2×10 3 μmol): (5×10 3 ~1.5×10 4 μmol): (5×10 2 ~2×10 3 μmol); (50~200μmol).

4. A culture system, characterized in that, include: Culture medium and the culture composition according to any one of claims 1 to 3.

5. The culture system according to claim 4, characterized in that, This further includes transducing viruses; Optionally, the transducing virus is selected from at least one of Sendai virus, lentivirus, retrovirus, adenovirus, and adeno-associated virus.

6. The culture system according to claim 4, characterized in that, The culture media include RPMI 1640 medium, DMEM medium, F12 medium, and StemSpan. TM At least one of the SFEMII medium.

7. The culture system according to claim 4, characterized in that, The concentration of IL-6 in the culture system was 15–25 ng / mL; Optionally, the concentration of IL-3 in the culture system is 15–25 ng / mL; Optionally, the concentration of IL-15 in the culture system is 5–15 ng / mL; Optionally, the concentration of IL-2 in the culture system is 5–10 ng / mL; Optionally, the concentration of SCF in the culture system is 90–110 ng / mL; Optionally, the concentration of Flt3L in the culture system is 90–110 ng / mL; Optionally, the concentration of 740Y-P in the culture system is 0.5–2 μM; Optionally, the concentration of butyramide in the culture system is 0.05–0.2 μM; Optionally, the concentration of LBH589 in the culture system is 1–10 nM; Optionally, the concentration of LY2090314 in the culture system is 0.01–5 μM; Optionally, the concentration of Selumetinib in the culture system is 1–20 μM.

8. A method for reprogramming peripheral blood mononuclear cells, characterized in that, include: Peripheral blood mononuclear cells were cultured using the culture system described in any one of claims 4 to 7.

9. The method according to claim 8, characterized in that, The culture treatment was carried out in the following manner: Peripheral blood mononuclear cells were pre-cultured in a culture medium containing the cytokines and small molecule additives; We used Sendai virus and small molecule drugs to transduce pre-cultured peripheral blood mononuclear cells.

10. The method according to claim 8, characterized in that, The peripheral blood mononuclear cells were obtained by gradient centrifugation. Optionally, the culture treatment time is 20 to 28 hours.

11. The method according to claim 9, characterized in that, The cytokines include at least one of IL-6, IL-3, IL-15, IL-2, SCF, and Flt3L; Optionally, the cytokines include IL-6, IL-3, IL-15, IL-2, SCF, and Flt3L, and the small molecule drug includes LBH589; Optionally, the cytokines include IL-6, IL-3, IL-15, IL-2, SCF, and Flt3L, and the small molecule drug includes LY2090314; Optionally, the cytokines include IL-6, IL-3, IL-15, IL-2, SCF, and Flt3L, and the small molecule drug includes Selumetinib; Optionally, the molar ratio of IL-6, IL-3, IL-15, IL-2, SCF, Flt3L to LBH589 is (18–22 ng): (18–22 ng): (5–15 ng): (5–10 ng): (90–110 ng): (90–110 ng): (2 × 10⁻⁶ ng) 3 ~1×10 4 nmol); Optionally, the molar ratio of IL-6, IL-3, IL-15, IL-2, SCF, Flt3L to LY2090314 is (18–22 ng): (18–22 ng): (5–15 ng): (5–10 ng): (90–110 ng): (90–110 ng): (5 × 10⁻⁶ ng) 2 ~2×10 3 μmol); Optionally, the molar ratio of IL-6, IL-3, IL-15, IL-2, SCF, Flt3L to Selumetinib is (18–22 ng): (18–22 ng): (5–15 ng): (5–10 ng): (90–110 ng): (90–110 ng): (5 × 10⁻⁶ ng) 3 ~1.5×10 4 μmol); Optionally, the molar ratio of IL-6, IL-3, IL-15, IL-2, SCF, Flt3L, LBH589, LY2090314, and Selumetinib is (18–22 ng): (18–22 ng): (5–15 ng): (5–10 ng): (90–110 ng): (90–110 ng): (2 × 10⁻⁶ ng) 3 ~1×10 4 nmol): (5×10 2 ~2×10 3 μmol): (5×10 3 ~1.5×10 4 μmol); Optionally, the molar ratio of the IL-6, IL-3, IL-15, IL-2, SCF, Flt3L, LBH589, LY2090314, Selumetinib, 740Y-P, and butyramide is (18–22 ng): (18–22 ng): (5–15 ng): (5–10 ng): (90–110 ng): (90–110 ng): (2 × 10⁻⁶ ng) 3 ~1×10 4 nmol): (5×10 2 ~2×10 3 μmol): (5×10 3 ~1.5×10 4 μmol): (5×10 2 ~2×10 3 μmol); (50~200μmol).

12. The method according to claim 9, characterized in that, The concentration of IL-6 in the culture medium was 15–25 ng / mL; Optionally, the concentration of IL-3 in the culture medium is 15–25 ng / mL; Optionally, the concentration of IL-15 in the culture medium is 5–15 ng / mL; Optionally, the concentration of IL-2 in the culture medium is 5–10 ng / mL; Optionally, the concentration of SCF in the culture medium is 90–110 ng / mL; Optionally, the concentration of Flt3L in the culture medium is 90–110 ng / mL; Optionally, the concentration of 740Y-P in the culture medium is 0.5–2 μM; Optionally, the concentration of butyramide in the culture medium is 0.05–0.2 μM; Optionally, the concentration of LBH589 in the culture medium is 1–10 nM; Optionally, the concentration of LY2090314 in the culture medium is 0.01–5 μM; Optionally, the concentration of Selumetinib in the culture medium is 1–20 μM.

13. A method for detecting the reprogramming efficiency of peripheral blood mononuclear cells, characterized in that, include: Peripheral blood mononuclear cells are reprogrammed using the method described in any one of claims 8 to 12; Pluripotency markers were detected in reprogrammed cells.

14. An induced pluripotent stem cell, characterized in that, Obtained by the method described in any one of claims 8 to 12.

15. The application of the induced pluripotent stem cells according to claim 14 in regenerative medicine.

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