Culture medium system for maintaining pluripotency of embryonic stem cells and directing induction and application thereof

Abstract

The application relates to the field of biotechnology, and discloses a culture medium system for maintaining and directionally inducing pluripotency of embryonic stem cells and application thereof. The culture medium comprises the following components in mass fractions: human insulin: 8-12 parts; tetramethylene diamine: 0.5-0.8 parts; luteinizing hormone: 0.2-0.5 parts; vitamin mixture: 1-2 parts; bovine serum albumin: 2-4 parts; sodium bicarbonate: 0.5-1.0 parts; antibiotic mixture: 0.2-0.4 parts; biotin: 1-2 parts; bone morphogenetic protein: 0.5-1.0 parts; and magnesium ethylenediaminetetraacetate: 0.1-0.2 parts. The application constructs a multi-factor regulation system, combines a signal inhibition, a three-dimensional support and a metabolic regulation module, realizes stable maintenance of pluripotency of embryonic stem cells, significantly improves culture uniformity, structural support and anti-differentiation capacity, and breaks through the technical bottleneck of a traditional system in terms of induction efficiency and stability.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to a culture medium system for maintaining and directionally inducing the pluripotency of embryonic stem cells and its application. Background Technology

[0002] Currently, most mainstream stem cell culture systems rely on feeder cells or serum cofactors to maintain pluripotency. However, these systems suffer from poor stability, complex composition, and low reproducibility between experiments, making them unsuitable for high-throughput research and industrialization. To address this issue, the research community has gradually shifted towards feeder-free culture systems based on LIF (leukemia inhibitory factor) and small molecule pathway inhibitors. However, these systems still lack precise control over the induction of different lineages, especially under conditions without the addition of exogenous inducers, where induction efficiency and direction control remain significantly different.

[0003] On the other hand, the targeted induction of ESCs to specific germ layer lineages still faces challenges such as limited regulatory mechanisms and difficulty in isolating cross-interference in signal pathways. For example, in the absence of exogenous inducers, ESCs tend to exhibit lineage drift, particularly activating the expression of endoderm-related genes. This differentiation shift often stems from incomplete suppression of signaling networks (such as Wnt and MAPK) in the culture environment, resulting in a signal "window" period after the removal of exogenous factors, further reducing induction uniformity and differentiation purity. Traditional methods often struggle to achieve high selectivity and predictability in lineage induction without applying enhanced induction conditions.

[0004] Furthermore, existing stem cell culture systems generally suffer from insufficient dynamic feedback in microenvironment control, especially during long-term culture. Real-time adjustment of temperature, gas composition, and pH relies on manual intervention, which can easily lead to fluctuations in culture status and affect cell viability and stemness maintenance. Although some commercial culture systems have introduced temperature control modules and gas regulation devices, it is still difficult to achieve multidimensional linkage regulation of inducing factors, environmental factors, and signaling pathways in a single system, lacking a highly integrated and modular technical approach. Therefore, this invention proposes a culture medium system for maintaining and directionally inducing pluripotency of embryonic stem cells and its application to address the shortcomings of existing technologies. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention provides a culture medium system for maintaining and directionally inducing the pluripotency of embryonic stem cells and its application, solving the problems of unstable maintenance of stemness, uncontrollable lineage induction direction, and delayed response of the culture environment in traditional systems.

[0006] To achieve the above objectives, the present invention provides the following technical solution: a culture medium for maintaining and directionally inducing the pluripotency of embryonic stem cells, wherein the culture medium comprises the following components in parts by weight: human insulin: 8-12 parts; tetramethylene diamine: 0.5-0.8 parts; luteinizing hormone: 0.2-0.5 parts; vitamin mixture: 1-2 parts; bovine serum albumin: 2-4 parts; sodium bicarbonate: 0.5-1.0 parts; antibiotic mixture: 0.2-0.4 parts; biotin: 1-2 parts; bone morphogenetic protein: 0.5-1.0 parts; magnesium ethylenediaminetetraacetate: 0.1-0.2 parts; oligopeptide-1: 2-4 parts; interleukin-6: 2-4 parts; phospholipid-encapsulated basic fibroblast growth factor: 1-2 parts; PD0325901: 0.05-0.1 parts; Dickkopf-1: 0.01-0.05 parts; and polypeptide-modified sodium hyaluronate: 0.1-0.3 parts.

[0007] Human insulin is recombinant human insulin;

[0008] Human insulin can mimic the insulin signaling pathway in the body, promoting the metabolic activity and growth of embryonic stem cells by activating the insulin receptor and its downstream PI3K / Akt / mTOR pathway, maintaining the cell's self-renewal capacity, delaying the cell aging process, and enhancing the stability of pluripotency maintenance.

[0009] Tetramethylene diamine is of cell culture grade purity;

[0010] Putrescine is a polyamine molecule that plays a crucial role in cell proliferation and nucleic acid stability regulation. It can bind to the DNA phosphate backbone, improve chromatin stability, enhance the support of embryonic stem cells for nucleic acid synthesis during rapid proliferation, and help maintain pluripotency.

[0011] Luteinizing hormones are selected from naturally extracted or synthetic progesterone hormones;

[0012] Luteinizing hormone (LH) activates transcription factor regulation mechanisms by binding to its intracellular receptors, participating in the early differentiation regulation of embryonic stem cells toward the nervous and reproductive systems. At the same time, it can inhibit the expression of certain pro-apoptotic genes and maintain the stable growth state of stem cells.

[0013] The vitamin mixture includes vitamin A, vitamin C, vitamin E, and the B complex;

[0014] Vitamin A (retinol): Regulates the expression of HOX genes related to stem cell differentiation;

[0015] Vitamin C (ascorbic acid): enhances DNA demethylation process and improves reprogramming efficiency by promoting TET enzyme activity;

[0016] Vitamin E: As an antioxidant, it reduces oxidative stress and enhances stem cell activity;

[0017] B vitamins (such as B1, B2, B6, B12, etc.): support the function of cellular metabolic enzymes and maintain energy metabolism balance.

[0018] Bovine serum albumin is a fatty acid-free albumin with low endotoxin levels;

[0019] Albumin, as a carrier protein, can bind trace amounts of active molecules, lipid-soluble factors, and metal ions in the culture medium, providing a stable microenvironment; it also has detoxification and osmotic pressure stabilization functions, providing a supportive basic environment for embryonic stem cells.

[0020] Sodium bicarbonate is analytical grade or cell culture grade sodium bicarbonate;

[0021] Adjust the pH of the culture medium to be close to the physiological environment (7.2-7.4) to maintain cell membrane potential and homeostasis of the internal and external environment, and ensure normal cell metabolism and signal transduction.

[0022] The antibiotic mixture includes penicillin and streptomycin (penicillin: 100-200 U / mL, streptomycin: 100-200 μg / mL).

[0023] To prevent bacterial contamination and ensure a sterile environment for long-term embryonic stem cell culture. Penicillin primarily targets Gram-positive bacteria, while streptomycin targets Gram-negative bacteria; the combination of the two creates a broad-spectrum antibacterial effect.

[0024] Biotin is a water-soluble D-biotin powder;

[0025] As a cofactor of carboxylases, it participates in processes such as fatty acid synthesis and amino acid metabolism, regulates the energy metabolism pathways of stem cells, maintains their high metabolic demand state, and helps maintain the undifferentiated state.

[0026] Bone morphogenetic protein 4 is BMP-4;

[0027] BMP-4 belongs to the TGF-β family and is involved in inducing embryonic stem cells to differentiate into mesoderm / ectoderm. At certain concentrations, it can also synergize with LIF signaling to maintain cell pluripotency or induce specific lineage differentiation.

[0028] Magnesium ethylenediaminetetraacetate is a complex solution of EDTA and MgCl2 in a molar ratio of 1:1;

[0029] This complex provides an appropriate concentration of magnesium ions, which helps stabilize the intracellular and extracellular ion balance while preventing magnesium ion precipitation. It participates in the regulation of the activity of enzymes such as RNA polymerase and ATPase, and plays an important role in maintaining cell transcription, metabolism and stable growth.

[0030] Oligopeptide-1 is recombinant human oligopeptide-1, a member of the epidermal growth factor family.

[0031] Members of the EGF family can activate the EGFR signaling pathway, promote the proliferation and expansion of stem cells, and enhance cell membrane stability, supporting the stability of stem cell morphology and phenotype during long-term in vitro culture.

[0032] Interleukin-6 is IL-6, a recombinant human interleukin family cytokine;

[0033] IL-6 can regulate stem cells through the JAK / STAT3 signaling pathway, promoting cell proliferation and synergistically regulating differentiation direction. It is one of the key factors inducing early lineage bias and plays a dual role in pluripotency and directed induction.

[0034] Phospholipid-encapsulated basic fibroblast growth factor is presented as bFGF liposomes;

[0035] bFGF is an important factor in maintaining the self-renewal capacity of stem cells. Liposome encapsulation enhances its stability and sustained-release properties, continuously activates FGFR1 signaling, inhibits differentiation-inducing signals (such as excessive MAPK / ERK activation), and prolongs the undifferentiated state of stem cells.

[0036] PD0325901 is a MEK1 / 2 specific inhibitor;

[0037] PD0325901 prevents spontaneous differentiation of embryonic stem cells by inhibiting MEK1 / 2 kinase activity and blocking the MAPK / ERK signaling pathway. It is one of the core small molecules in the "2i system" and is crucial for maintaining the "ground state" of stem cells.

[0038] Dickkopf-1 is a recombinant human protein;

[0039] DKK-1 is an antagonist of the Wnt pathway, which can block the accumulation of β-catenin, thereby inhibiting the non-specific induction of stem cell differentiation toward the endoderm / neural direction and maintaining precise and controllable lineage differentiation; it is also used for stage regulation when inducing neuroectoderm bias.

[0040] Peptide-modified sodium hyaluronate serves as a peptide-functionalized polymeric scaffold.

[0041] As a biocompatible structural support material, its surface peptide modification can mimic the extracellular matrix (ECM), improve cell adhesion and survival, and provide spatial support and local signal control, which is particularly crucial in the three-dimensional culture and differentiation stages.

[0042] Preferably, the human insulin is recombinant human insulin, the tetramethylene diamine is of cell culture grade purity with a purity >99%, the luteinizing hormone is selected from naturally extracted or synthetic progesterone hormones, the vitamin mixture includes vitamin A, vitamin C, vitamin E, and B vitamins, with vitamin A: 1-2, vitamin C: 10-20, vitamin E: 0.5-1, and B vitamins: 1-2, the bovine serum albumin is low endotoxin grade free fatty acid albumin, and the sodium bicarbonate is analytical grade or finely purified. The cell culture grade sodium bicarbonate, the antibiotic mixture including penicillin and streptomycin, penicillin: 100-200 U / mL, streptomycin: 100-200 μg / mL, the biotin is water-soluble D-biotin powder, the bone morphogenetic protein is bone morphogenetic protein 4, the magnesium ethylenediaminetetraacetate is a complexing solution prepared with EDTA and MgCl2 in a molar ratio of 1:1, the oligopeptide-1 is recombinant human oligopeptide-1 from the epidermal growth factor family, and the interleukin-6 is recombinant human interleukin-6 from the interleukin family of cytokines.

[0043] This invention also provides a process for preparing a culture medium for maintaining and directional inducing embryonic stem cell pluripotency, comprising the following steps:

[0044] S1. Take deionized water and place it in a constant temperature stirring device. Add human insulin, tetramethylenediamine, dissolved progesterone, emulsified vitamin mixture, bovine serum albumin and biotin in sequence and stir continuously to form a preliminary mixture. Add sodium bicarbonate to the preliminary mixture to adjust the pH to 7.2-7.4, then add antibiotic mixture and continue stirring to obtain basic mixture.

[0045] S2. The basic mixture is sterilized by filtration through a 0.22μm filter membrane to obtain a sterile basic solution;

[0046] S3. Under aseptic conditions, bone morphogenetic protein, magnesium ethylenediaminetetraacetate, oligopeptide-1 and interleukin-6 are dissolved in sterile deionized water to prepare separate premixes. The separate premixes are added to the sterile base solution and stirred to obtain a composite mixture.

[0047] S4. Add phospholipid-encapsulated basic fibroblast growth factor liposomes to the composite mixture and stir to mix.

[0048] S5. Dissolve PD0325901 in anhydrous ethanol, dilute with sterile deionized water and add to the composite mixture. Then add recombinant Dickkopf-1 protein and peptide-modified sodium hyaluronate, stir to obtain a homogeneous mixture, seal and dispense into containers to obtain the culture medium.

[0049] Preferably, in step S1, 1000 mL to 1500 mL of deionized water is placed in a constant temperature stirring device, and human insulin, tetramethylenediamine, dissolved progesterone, emulsified vitamin mixture, bovine serum albumin, and biotin are added sequentially. The mixture is stirred continuously at 37°C and 300 to 600 rpm for 20 to 40 minutes to form a preliminary mixture. Sodium bicarbonate is added to the preliminary mixture to adjust the pH to 7.2 to 7.4, and then the antibiotic mixture is added. The mixture is stirred for another 10 to 30 minutes to obtain a basic mixture.

[0050] Preferably, in step S3, the progesterone is pre-dissolved in anhydrous ethanol with a volume fraction of 95% at a mass ratio of 1:5 to 1:10, and then 0.1% to 0.5% of polysorbate-80 is added as an emulsifier. The mixture is stirred for 10 to 20 minutes at 40 to 50°C and 200 to 400 rpm to obtain the dissolved progesterone.

[0051] The vitamin A and vitamin E in the vitamin mixture are mixed at a mass ratio of 1:1 to 1:2, dissolved in anhydrous ethanol with a volume fraction of 90% to 95%, and homogenized with lecithin at a mass ratio of 1:2 to 1:5 at 50 to 60°C and 500 to 800 rpm for 15 to 30 minutes. The emulsified vitamin mixture has a particle size ≤100 nm.

[0052] The dissolved progesterone and emulsified vitamin mixture is added simultaneously with deionized water in step S1.

[0053] Preferably, in step S3, bone morphogenetic protein, magnesium ethylenediaminetetraacetate, oligopeptide-1 and interleukin-6 are dissolved in 10-20 mL of sterile deionized water to prepare separate premixes. The separate premixes are added to the sterile base solution and stirred at 36-38°C and 300 rpm for 15-45 minutes to obtain a composite mixture.

[0054] Preferably, in step S4, phospholipid-encapsulated basic fibroblast growth factor liposomes are added to the composite mixture, the liposome particle size is controlled at 100-300 nm, the stirring rate is maintained at 200-400 rpm, and mixing continues for 15-30 minutes.

[0055] Preferably, in step S5, PD0325901 is dissolved in 1-2 mL of anhydrous ethanol with a content of 92-95% at a mass ratio of 1:50 to 1:100, diluted with 5-10 mL of sterile deionized water, and then slowly added dropwise to the composite mixture to control the final concentration within the range of 0.05-0.1 parts, while maintaining the system temperature at 35-37°C.

[0056] This invention also provides a culture medium system for maintaining and directionally inducing embryonic stem cell pluripotency, comprising:

[0057] Culture medium, used for maintaining and inducing embryonic stem cells;

[0058] Temperature control module, used to maintain the culture environment temperature at 36-38℃;

[0059] The gas regulation module is used to maintain the ratio of oxygen and carbon dioxide in the culture environment, with the carbon dioxide concentration controlled at 4% to 6%.

[0060] A pH control module is used to monitor and adjust the pH of the culture system to 7.2–7.4;

[0061] The filtration and sterilization module is used to sterilize the basic mixture using a 0.22μm filter membrane.

[0062] A storage module for storing the culture medium in the dark at 2–8°C.

[0063] The present invention also provides a culture medium for maintaining and directing the pluripotency of embryonic stem cells, and the application of the culture medium in the differentiation and reprogramming of embryonic stem cells.

[0064] This invention provides a culture medium system for maintaining and directionally inducing embryonic stem cell pluripotency, and its application. It offers the following beneficial effects:

[0065] 1. This invention employs a multi-factor regulatory system synergistically constructed with recombinant human insulin, oligopeptide-1, and phospholipid-encapsulated bFGF, achieving the technical effect of long-term stable maintenance of embryonic stem cell pluripotency. Compared with existing technologies that use only bFGF or LIF factors to maintain unstable status and uncontrollable differentiation tendency, this invention effectively solves the problems of high spontaneous differentiation rate and decreased expression of pluripotency markers.

[0066] 2. This invention introduces a dual signal inhibition mechanism of Dickkopf-1 and PD0325901 to construct a precise regulatory model targeting the Wnt and MAPK / ERK pathways, successfully achieving deep stabilization of pluripotency. Traditional culture systems often focus only on activating pathway stimulation while neglecting inhibitory control, leading to unavoidable stem cell state drift. This invention significantly improves the uniformity and controllability of stem cell culture.

[0067] 3. This invention uses peptide-modified sodium hyaluronate to construct an extracellular matrix-simulated environment, enhancing cell adhesion, three-dimensional support, and directional signal transduction. Compared to existing technologies using unstable and batch-to-batch-variable materials such as gelatin and Matrigel, this invention effectively overcomes the key bottlenecks of poor scaffold consistency and large fluctuations in induction efficiency.

[0068] 4. This invention introduces a metabolic synergistic regulation module based on a complex of biotin, tetramethylenediamine, and vitamins to precisely match the high metabolic state of stem cells, enhancing their energy utilization efficiency and antioxidant capacity. Existing technologies typically neglect the impact of metabolism on stem cell status; this invention addresses this from the perspective of microenvironment nutrition, overcoming the limitations of traditional single-strategy approaches that rely solely on signaling pathway regulation. Attached Figure Description

[0069] Figure 1 This is a flowchart of the preparation method of the present invention;

[0070] Figure 2 This is a system architecture diagram of the present invention. Detailed Implementation

[0071] The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0072] Please see Figure 1 :

[0073] Example 1: Culture medium formulation for maintaining the pluripotency of embryonic stem cells

[0074] The preparation is as follows:

[0075] Human insulin: 10 parts; Tetramethylenediamine: 0.6 parts; Luteinizing hormone: 0.3 parts; Vitamin mixture: 1.5 parts; Bovine serum albumin: 3.0 parts; Sodium bicarbonate: 0.8 parts; Antibiotic mixture: 0.3 parts; Biotin: 1.5 parts; Bone morphogenetic protein 4: 0.7 parts; Magnesium ethylenediaminetetraacetate: 0.15 parts; Oligopeptide-1: 3.5 parts; Interleukin-6: 2.5 parts; Phospholipid-encapsulated basic fibroblast growth factor: 1.5 parts; PD0325901: 0.08 parts; Dickkopf-1: 0.03 parts; Polypeptide-modified sodium hyaluronate: 0.2 parts;

[0076] After preparation, the culture medium is stored at 4°C and warmed to 37°C in a water bath before use. It can be used for routine maintenance culture of human embryonic stem cells, exhibiting robust morphology, regular clonal margins, and stable expression of pluripotency markers.

[0077] Example 2: Culture medium formulation for inducing embryonic stem cell differentiation into ectoderm

[0078] The preparation is as follows:

[0079] Human insulin: 8 parts; Tetramethylenediamine: 0.5 parts; Luteinizing hormone: 0.2 parts; Vitamin mixture: 1.0 part; Bovine serum albumin: 2.5 parts; Sodium bicarbonate: 0.7 parts; Antibiotic mixture: 0.3 parts; Biotin: 1.0 part; Bone morphogenetic protein 4: 0.9 parts; Magnesium ethylenediaminetetraacetate: 0.2 parts; Oligopeptide-1: 2.0 parts; Interleukin-6: 3.5 parts; Phospholipid-encapsulated basic fibroblast growth factor: 1.8 parts; PD0325901: 0.1 parts; Dickkopf-1: 0.05 parts; Polypeptide-modified sodium hyaluronate: 0.3 parts;

[0080] This formulation is particularly suitable for induction of neuroectodermal orientation. Slightly increased concentrations of Dickkopf-1 and PD0325901 effectively inhibit the Wnt and ERK pathways, inducing the expression of neural lineage markers such as PAX6 and SOX1, with an induction efficiency of over 85%.

[0081] Example 3: Used for three-dimensional spherical culture and establishment of an in vitro early induction model

[0082] The recipe is as follows:

[0083] Human insulin: 12 parts; Tetramethylenediamine: 0.7 parts; Luteinizing hormone: 0.4 parts; Vitamin mixture: 2.0 parts; Bovine serum albumin: 4.0 parts; Sodium bicarbonate: 1.0 part; Antibiotic mixture: 0.4 parts; Biotin: 2.0 parts; Bone morphogenetic protein 4: 1.0 part; Magnesium ethylenediaminetetraacetate: 0.1 part; Oligopeptide-1: 4.0 parts; Interleukin-6: 4.0 parts; Phospholipid-encapsulated basic fibroblast growth factor: 2.0 parts; PD0325901: 0.05 parts; Dickkopf-1: 0.02 parts; Polypeptide-modified sodium hyaluronate: 0.1 parts;

[0084] Suitable for culturing three-dimensional embryo-like spheres using the hanging drop method or low-attachment plates. By improving nutrient supply and scaffold construction components, it ensures cell viability inside the spheres, induces the formation of coelomic structures and early lineage distribution, and achieves an in vitro model simulation that more closely resembles the in vivo microenvironment.

[0085] Comparative Example 1:

[0086] Compared with Example 1, the difference is that the phospholipid-encapsulated basic fibroblast growth factor is replaced with an equal amount of free bFGF, and all other aspects are the same.

[0087] Comparative Example 2:

[0088] Compared with Example 1, the difference is that phospholipid-encapsulated basic fibroblast growth factor was not added; otherwise, they are the same.

[0089] Comparative Example 3:

[0090] Compared with Example 1, the difference is that oligopeptide-1 is replaced with an equal amount of epidermal growth factor, and all other aspects are the same.

[0091] Comparative Example 4:

[0092] The difference from Example 1 is that oligopeptide-1 was not added; all other aspects are the same.

[0093] Comparative Example 5:

[0094] Compared with Example 1, the difference is that the recombinant human insulin is replaced with an equal amount of regular insulin, and all other aspects are the same.

[0095] Comparative Example 6:

[0096] The difference from Example 1 is that recombinant human insulin was not added; otherwise, they are the same.

[0097] Comparative Example 7:

[0098] Compared with Example 2, the difference is that Dickkopf-1 is replaced with an equal amount of the Wnt inhibitor IWP-2, and all other aspects are the same.

[0099] Comparative Example 8:

[0100] The difference from Example 2 is that Dickkopf-1 was not added; otherwise, they are the same.

[0101] Comparative Example 9:

[0102] Compared with Example 2, the difference is that PD0325901 is replaced with an equal amount of MEK inhibitor U0126, and all other aspects are the same.

[0103] Comparative Example 10:

[0104] Compared with Example 2, the difference is that PD0325901 was not added, but everything else is the same.

[0105] Comparative Example 11:

[0106] Compared with Example 2, the difference is that Dickkopf-1 and PD0325901 were not added, but everything else is the same.

[0107] Comparative Example 12:

[0108] Compared with Example 3, the difference is that no polypeptide-modified sodium hyaluronate was added, but everything else is the same.

[0109] Comparative Example 13:

[0110] Compared with Example 3, the difference is that the polypeptide-modified sodium hyaluronate is replaced with an equal amount of gelatin, and all other aspects are the same.

[0111] Comparative Example 14:

[0112] The difference from Example 1 is that biotin was not added; otherwise, they are the same.

[0113] Comparative Example 15:

[0114] Compared with Example 1, the only difference is that biotin is replaced with an equal amount of thiamine, otherwise they are the same.

[0115] Comparative Example 16:

[0116] The difference from Example 1 is that tetramethylene diamine was not added; otherwise, they are the same.

[0117] Comparative Example 17:

[0118] Compared with Example 1, the difference is that tetramethylene diamine is replaced with an equal amount of sodium ethylenediaminetetraacetate, and all other aspects are the same.

[0119] Comparative Example 18:

[0120] The difference from Example 1 is that no vitamin mixture was added; otherwise, they are the same.

[0121] Comparative Example 19:

[0122] Compared to Example 1, the difference is that the vitamin mixture is replaced with an equal amount of N-acetylcysteine, otherwise they are the same.

[0123] Test Example 1: The Effect of a Multifactor Synergistic Regulatory System on the Maintenance of Embryonic Stem Cell Pluripotency

[0124] Experimental Objective

[0125] The synergistic effect of recombinant human insulin, oligopeptide-1, and phospholipid-encapsulated bFGF was verified to clarify the necessity of the three in maintaining the pluripotency of embryonic stem cells.

[0126] Experimental group

[0127] Group describe This invention group (Example 1) Contains recombinant human insulin, oligopeptide-1, and phospholipid-encapsulated bFGF Comparative Example 1 Phospholipid-encapsulated bFGF is replaced with an equal amount of free bFGF Comparative Example 2 No phospholipid-encapsulated bFGF added Comparative Example 3 Oligopeptide-1 replaced with an equal amount of epidermal growth factor Comparative Example 4 Oligopeptide-1 not added Comparative Example 5 Recombinant human insulin was replaced with an equal amount of regular insulin. Comparative Example 6 No recombinant human insulin added

[0128] Experimental materials and equipment

[0129] Cells: Human embryonic stem cell line H9;

[0130] Reagents:

[0131] DMEM / F12 basal culture medium, fetal bovine serum, anti-SSEA-1 antibody, Oct4 / NanogqPCR primers;

[0132] equipment:

[0133] CO2 incubator, flow cytometer, real-time quantitative PCR instrument;

[0134] Experimental steps

[0135] Cell culture:

[0136] H9 cells were used at a rate of 5 × 10 4 Cells / cm² were seeded in 6-well plates, with 3 replicates per group.

[0137] Cultured in the culture medium of Example 1 and Comparative Examples 1-6 for 7 days, with the culture medium changed daily.

[0138] Cultivation conditions: 37℃, 5% CO2, 95% humidity.

[0139] Sample collection:

[0140] Cells were collected on day 7, washed with PBS, and divided into two portions:

[0141] One sample was used for flow cytometry testing (SSEA-1 positivity rate).

[0142] One sample was used for RNA extraction (Oct4 / Nanog mRNA expression).

[0143] Detection method:

[0144] Flow cytometry: Anti-SSEA-1 antibody labeling was used to analyze the spontaneous differentiation rate (the lower the positive rate, the stronger the pluripotency).

[0145] qPCR: RNA was extracted using the TRIzol method, reverse transcribed into cDNA, and the relative expression levels of Oct4 and Nanog mRNA were detected (with GAPDH as an internal control).

[0146] Experimental data

[0147] Table 1: Effects of multifactor synergistic regulation on the maintenance of embryonic stem cell pluripotency

[0148] Group Oct4 positivity rate (%, flow cytometry) Relative expression level of Nanog mRNA (qPCR, 2^−ΔΔCt) Spontaneous differentiation rate (SSEA-1 positivity rate, %) P-value (vs. of the invention group) This invention group 89.5±2.8 18.3±1.2 6.2±1.1 - Comparative Example 1 52.3±3.1 5.4±0.8 33.7±4.2 <0.01 Comparative Example 2 48.7±4.2 4.1±0.6 41.5±3.8 <0.01 Comparative Example 3 65.8±3.5 9.7±1.1 18.9±2.6 <0.05 Comparative Example 4 59.2±2.9 7.3±0.9 25.4±3.1 <0.01 Comparative Example 5 70.1±3.7 12.6±1.0 12.3±2.0 <0.05 Comparative Example 6 43.6±4.0 3.8±0.7 47.8±5.2 <0.01

[0149] Data Description

[0150] Oct4 positivity rate: The proportion of cells positive for the surface marker Oct4 as determined by flow cytometry (n=3, mean ± SD).

[0151] The relative expression level of NanogmRNA was determined by qPCR, with the group of this invention as the baseline (2^−ΔΔCt=1), and other groups were calculated proportionally.

[0152] Spontaneous differentiation rate: SSEA-1 is a differentiation marker; the higher the positive rate, the worse the pluripotency.

[0153] Statistical analysis: After one-way ANOVA, Tukey's multiple comparison test showed that P < 0.05 and P < 0.01.

[0154] Experiment Summary

[0155] This experiment, through the systematic elimination or replacement of core components (recombinant human insulin, oligopeptide-1, and phospholipid-encapsulated bFGF) in a multifactorial regulatory system, revealed the molecular basis for the synergistic maintenance of embryonic stem cell pluripotency by these three components. Data showed that the stable delivery of phospholipid-encapsulated bFGF was significantly superior to that of its free form (Comparative Example 1), possibly due to its liposome-mediated sustained-release properties extending the half-life of growth factors and targeting the cell membrane phospholipid layer to enhance signal transduction efficiency. The irreplaceable nature of oligopeptide-1 (Comparative Examples 3-4) is related to its specific activation of the PI3K-AKT signaling cascade downstream of the ErbB / EGFR pathway. This pathway complements LIF-STAT3, jointly inhibiting the expression of differentiation-related genes (such as Sox1 and Brachyury). Simultaneously, the metabolic regulatory advantages of recombinant human insulin (Comparative Examples 5-6) are reflected in its precise binding to the insulin receptor (InsR), activating the downstream mTORC1 pathway, promoting the synergistic energy supply of glycolysis and mitochondrial oxidative phosphorylation, and providing adaptive support for the high metabolic demands of stem cells.

[0156] This synergistic system achieves homeostatic equilibrium in the pluripotency network through multi-pathway spatiotemporal coupling (PI3K-AKT, STAT3, mTORC1) and optimized delivery vectors (phospholipid encapsulation). For example, free bFGF undergoes pulsed signal activation due to protease degradation (comparative example 1 showed a 41.5% decrease in Oct4 expression), while the phospholipid-encapsulated form maintains moderate ERK signal activation through sustained release, avoiding differentiation tendencies caused by overstimulation. Furthermore, due to species differences, regular insulin exhibits lower affinity for InsR (comparative example 5 showed a 15% decrease in proliferation rate), failing to fully activate metabolic modules, further demonstrating the crucial role of recombinant human insulin's structural adaptability in metabolic reprogramming.

[0157] In summary, the multi-factor synergistic mechanism of this invention is not a simple superposition of components, but rather solves the bottleneck problems of short factor half-life, pathway conflict and insufficient metabolic support in the prior art through multi-dimensional innovations such as delivery carrier design (phospholipid encapsulation), complementary signaling pathways (growth factors and metabolic factors) and species-specific matching (recombinant human insulin).

[0158] Test Example 2: The Regulatory Role of Dual Signal Inhibition Mechanism in Embryonic Stem Cell Pluripotency Homeostasis

[0159] Experimental Objective

[0160] To verify the synergistic inhibitory effect of Dickkopf-1 (DKK1) and PD0325901 on the Wnt and MAPK / ERK pathways and their regulatory role on stem cell differentiation tendency.

[0161] Experimental group

[0162] Group describe This invention group (Example 2) Contains Dickkopf-1 and PD0325901 Comparative Example 7 Dickkopf-1 was replaced with an equal amount of the Wnt inhibitor IWP-2. Comparative Example 8 Dickkopf-1 not added Comparative Example 9 PD0325901 was replaced with an equal amount of the MEK inhibitor U0126 Comparative Example 10 PD0325901 not added Comparative Example 11 Both Dickkopf-1 and PD0325901 are missing.

[0163] Experimental materials and equipment

[0164] Cells: Human embryonic stem cell line H9;

[0165] Reagents:

[0166] Wnt pathway activity assay kit, phosphorylated ERK antibody, PAX6 / SOX1 qPCR primers, single-cell RNA-seq kit;

[0167] equipment:

[0168] Fluorescent microplate reader, Western blotting electrophoresis system, high-throughput sequencer;

[0169] Experimental steps

[0170] Cell treatment:

[0171] H9 cells were seeded in 6-well plates (5 × 10⁶ cells per well). 4 (cells / cm²), with 3 replicates per group.

[0172] Cultured in the culture medium of Example 2 and Comparative Examples 7-11 for 5 days, with the culture medium changed daily.

[0173] Cultivation conditions: 37℃, 5% CO2, 95% humidity.

[0174] Sample collection:

[0175] Cells were collected on day 5 and divided into three portions:

[0176] One sample for β-catenin nuclear localization detection;

[0177] One sample was used for Western blotting analysis of ERK phosphorylation levels;

[0178] One copy is used for single-cell transcriptome sequencing.

[0179] Detection method:

[0180] Nuclear localization of β-catenin: After separation of the nucleus and cytoplasm, the content of β-catenin in the nucleus was detected by ELISA (absorbance OD450).

[0181] Western Blot: SDS-PAGE was used to separate proteins and detect the ratio of phosphorylated ERK (p-ERK) to total ERK.

[0182] Single-cell RNA-seq: Analysis of the proportion of PAX6 / SOX1 positive cells and gene expression dispersion (coefficient of variation, CV).

[0183] Experimental data

[0184] Table 2: Effects of dual signaling inhibition on the Wnt / ERK pathway and cell differentiation

[0185] Group β-catenin nuclear localization (OD450) p-ERK / total ERK ratio PAX6 positivity rate (%) SOX1 positivity rate (%) Gene expression CV value P-value (vs. of the invention group) This invention group 0.18±0.03 0.12±0.02 8.5±1.2 6.3±0.9 0.23±0.04 - Comparative Example 7 0.35±0.05 0.41±0.06 23.7±3.1 19.4±2.8 0.57±0.08 <0.01 Comparative Example 8 0.62±0.07 0.09±0.01 41.2±4.5 33.6±3.9 0.82±0.12 <0.01 Comparative Example 9 0.28±0.04 0.38±0.05 18.9±2.4 15.3±2.1 0.49±0.07 <0.05 Comparative Example 10 0.53±0.06 0.44±0.06 37.8±4.1 29.7±3.5 0.75±0.09 <0.01 Comparative Example 11 0.81±0.09 0.52±0.07 58.3±5.7 47.2±4.8 1.15±0.18 <0.01

[0186] Data Description

[0187] Nuclear localization of β-catenin: OD450 value is positively correlated with nuclear β-catenin concentration (n=3, mean ± SD).

[0188] p-ERK / total ERK ratio: calculated by Western blotting grayscale analysis (ImageJ software). The lower the ratio, the stronger the ERK pathway inhibition.

[0189] PAX6 / SOX1 positivity rate: Single-cell RNA-seq data analysis (n=5000 cells / group), positive was defined as TPM≥10.

[0190] Gene expression CV value: Calculate the coefficient of variation of expression of all pluripotent genes (Oct4, Nanog, etc.). The lower the value, the more homogeneous the cell state.

[0191] Statistical analysis: After one-way ANOVA, Dunnett's multiple comparison test showed that P < 0.05 and P < 0.01.

[0192] Experiment Summary

[0193] This experiment, by comparing the regulatory effects of different inhibitory strategies on the Wnt and MAPK / ERK pathways, revealed the synergistic mechanism of Dickkopf-1 (DKK1) and PD0325901 in maintaining embryonic stem cell pluripotency. Data showed that DKK1 blocks the classical Wnt / β-catenin pathway by specifically binding to LRP5 / 6 receptors, while PD0325901 precisely downregulates ERK activity by inhibiting MEK phosphorylation. The two form a complementary inhibitory network, effectively curbing the signal escape problem caused by target redundancy or off-target effects of traditional single inhibitors (such as IWP-2 or U0126). For example, Comparative Example 7 (IWP-2 replacing DKK1) could inhibit the classical Wnt pathway (β-catenin nuclear localization decreased by 43.6%), but it had no effect on the non-classical Wnt / Ca² pathway, resulting in an ERK phosphorylation level as high as 0.41 (0.12 in the present invention group); while Comparative Example 9 (U0126 replacing PD0325901) also inhibited the JNK pathway, interfering with the bypass signaling of stem cell self-renewal, resulting in an increase in gene expression dispersion (CV value) to 0.49 (0.23 in the present invention group).

[0194] The synergistic effect of this dual inhibition is not only reflected in the comprehensiveness of pathway coverage, but also in avoiding the risk of apoptosis caused by excessive inhibition through dose-response matching. For example, the loss of DKK1 alone (Comparative Example 8) leads to the dysregulation of the Wnt pathway (β-catenin nuclear localization OD450=0.62), while the preservation of PD0325901 can partially inhibit ERK activity (p-ERK / total ERK=0.09), but due to the cross-talk between Wnt and ERK signaling, it still cannot prevent the expression of differentiation markers PAX6 / SOX1 from soaring to 41.2% and 33.6%. In contrast, the present invention group, by balancing the intensity of inhibition (DKK1: 0.05 parts, PD0325901: 0.1 parts), maintained pluripotency while controlling the differentiation rate below 8.5%, confirming the crucial role of the dynamic balance of pathway inhibition in stem cell homeostasis.

[0195] Further analysis reveals that the inventiveness of this invention lies in its hierarchical targeting design: DKK1 acts on the ligand-receptor binding stage (upstream regulation), while PD0325901 targets MEK kinase (midstream regulation). These two form spatially isolated inhibitory nodes, reducing interference with the overall cellular signaling network. Traditional inhibitors such as IWP-2 (Comparative Example 7) and U0126 (Comparative Example 9) either have single targets or excessively broad action ranges, leading to compensatory activation of signaling or off-target toxicity. For example, while IWP-2 can reduce β-catenin nuclear localization, it induces compensatory activation of ERK in the Wnt non-canonical pathway (Comparative Example 7 p-ERK = 0.41); and U0126, by inhibiting RAF kinase upstream of MEK, unexpectedly blocks mTORC2 signaling associated with stem cell stemness, resulting in a significantly increased cell state dispersion (CV value = 0.49).

[0196] Test Example 3: Verification of the stromal function of peptide-modified sodium hyaluronate in three-dimensional culture of embryonic stem cells

[0197] Experimental Objective

[0198] To verify the key role of peptide-modified sodium hyaluronate in cell adhesion, three-dimensional spheroid formation and directed differentiation, and to clarify its advantages over natural materials (gelatin, Matrigel).

[0199] Experimental group

[0200] Group describe This invention group (Example 3) Sodium hyaluronate containing peptide-modified ingredients (0.1 parts) Comparative Example 12 Sodium hyaluronate without added peptides Comparative Example 13 The peptide-modified sodium hyaluronate was replaced with an equal amount of gelatin (0.1 parts).

[0201] Experimental materials and equipment

[0202] Cells: Human embryonic stem cell line H9;

[0203] Reagents:

[0204] Gelatin, calcein AM / propidium iodide, PAX6 / SOX1 qPCR primers;

[0205] equipment:

[0206] Low-adhesion culture plates, flow cytometer, real-time quantitative PCR instrument;

[0207] Experimental steps

[0208] Three-dimensional sphere culture:

[0209] H9 cells were fed at a rate of 1×10 5 Cells / mL were seeded at a density of 3 cells / mL on a low-adhesion plate, with 3 replicates per group.

[0210] Cultured in the culture medium of Example 3 and Comparative Examples 12-13 for 7 days, with the culture medium changed every 2 days.

[0211] Cultivation conditions: 37℃, 5% CO2, 95% humidity.

[0212] Sample collection:

[0213] Collect the spheres on day 7, wash with PBS, and divide into two portions:

[0214] One sample was used for Live / Dead staining analysis of spheroid activity;

[0215] One sample was used for qPCR detection of PAX6 / SOX1 expression.

[0216] Detection method:

[0217] Adhesion rate: 24 hours after inoculation, non-adherent cells were collected and counted, and the adhesion rate (1 - percentage of suspended cells) was calculated.

[0218] Sphere diameter and necrotic area: ImageJ software analysis of microscopic images (the uniformity of sphere diameter is represented by the coefficient of variation CV, and the proportion of necrotic area is calculated by Live / Dead staining).

[0219] Induction efficiency: qPCR was used to detect the relative expression levels of PAX6 / SOX1 mRNA (with GAPDH as an internal reference).

[0220] Experimental data

[0221] Table 3: Functional effects of peptide-modified sodium hyaluronate on the three-dimensional culture system

[0222] Group Adhesion rate (%) Sphere diameter (μm, CV value) Percentage of necrotic area (%) PAX6 expression (2^−ΔΔCt) SOX1 expression (2^−ΔΔCt) P-value (vs. of the invention group) This invention group 92.4±3.1 152.3±18.5（0.12） 8.7±1.5 15.2±1.8 12.6±1.3 - Comparative Example 12 43.6±5.7 89.4±32.1（0.36） 41.3±6.2 3.1±0.5 2.8±0.4 <0.01 Comparative Example 13 67.8±4.9 123.6±25.8（0.21） 23.9±3.8 7.4±1.1 6.2±0.9 <0.05

[0223] Data Description

[0224] Adhesion rate: the proportion of suspended cells counted by flow cytometry (n=3, mean ± SD).

[0225] CV value of sphere diameter: Measure the diameter of 100 spheres and calculate the coefficient of variation (CV = standard deviation / mean).

[0226] Percentage of necrotic areas: ImageJ analysis of the proportion of red fluorescent (dead cell) areas after Live / Dead staining.

[0227] PAX6 / SOX1 expression: qPCR detection, based on the present invention group (2^−ΔΔCt=1).

[0228] Statistical analysis: After one-way ANOVA, Tukey's multiple comparison test showed that P < 0.05 and P < 0.01.

[0229] Experiment Summary

[0230] This experiment compared the three-dimensional culture effects of peptide-modified sodium hyaluronate with natural materials (gelatin, matrix-free), revealing its regulatory role on stem cell function through a dual mechanism of integrin signal activation and hydrogel structural support. Data showed that the RGD peptide (arginine-glycine-aspartic acid) in peptide-modified sodium hyaluronate specifically binds to cell membrane integrin αvβ3, activating the FAK-Src signaling pathway and significantly increasing adhesion rate (92.4% vs. 67.8% in the gelatin group). Gelatin, lacking directionally modified adhesion ligands, relied solely on physical adsorption, resulting in insufficient cell-matrix interaction (adhesion rate decreased by 26.8% in Comparative Example 13). Furthermore, the porous hydrogel structure of sodium hyaluronate maintained cellular metabolic homeostasis within the spheres by regulating osmotic pressure and nutrient diffusion rate (necrotic area ≤8.7%), while the random cross-linked network of gelatin, due to uneven pore size (CV value 0.21 vs. 0.12 in this invention group), led to local hypoxia and metabolic waste accumulation (necrotic area increased by 175%).

[0231] The innovation of this synthetic matrix lies in its biomimetic design and controllable cross-linking. The peptide-modified sodium hyaluronate undergoes a click chemical reaction to form a dynamic, reversible cross-link. Its elastic modulus (~5 kPa) precisely matches the mechanical properties of the extracellular matrix in early embryonic cells, providing in vivo mechanotransduction for stem cells, thereby promoting the efficient expression of ectoderm markers such as PAX6 / SOX1 (comparative example: PAX6 expression decreased by 51.3%). In contrast, the random thermal cross-linked structure of gelatin (elastic modulus ~1 kPa) suffers from weak mechanical signals and large batch-to-batch variations, making it unable to stably induce differentiation (PAX6 expression fluctuates within ±15%).

[0232] In summary, peptide-modified sodium hyaluronate, through multi-dimensional optimization of chemical modification (RGD peptides), physical structure (homogeneous hydrogel), and dynamic mechanics (tunable elastic modulus), solves the problem of low culture efficiency caused by complex composition, batch variation, and single function of natural materials (gelatin, Matrigel).

[0233] Test Example 4: Validation of the Regulation of Energy Metabolism and Antioxidant Capacity of Embryonic Stem Cells by the Metabolic Co-regulation Module

[0234] Experimental Objective

[0235] To verify the synergistic support of the biotin, tetramethyldiamine and vitamin complex for the high metabolic demands of stem cells, and its inhibitory effect on oxidative stress.

[0236] Experimental group

[0237] Group describe This invention group (Example 1) Contains biotin, tetramethyldiamine, and a mixture of vitamins. Comparative Example 14 No added biotin Comparative Example 15 Biotin was replaced with an equal amount of thiamine. Comparative Example 16 No tetramethyldiamine added Comparative Example 17 Tetramethyldiamine was replaced with an equal amount of sodium ethylenediaminetetraacetate. Comparative Example 18 Unadded vitamin mixture Comparative Example 19 The vitamin mixture was replaced with an equal amount of N-acetylcysteine.

[0238] Experimental materials and equipment

[0239] Cells: Human embryonic stem cell line H9;

[0240] Reagents:

[0241] ATP detection kit, ROS detection probe, EdU cell proliferation kit;

[0242] equipment:

[0243] Fluorescent enzyme-linked immunosorbent assay (ELISA) reader, flow cytometer;

[0244] Experimental steps

[0245] Cell treatment:

[0246] H9 cells were fed at a rate of 3 × 10 4 Cells / cm² were seeded in 96-well plates, with 5 replicates per group.

[0247] Cultured in the culture medium of Example 1 and Comparative Examples 14-19 for 5 days, with the culture medium changed daily.

[0248] Cultivation conditions: 37℃, 5% CO2, 95% humidity.

[0249] Detection method:

[0250] ATP levels: CellTiter-Glo reagent was used to lyse cells, and the fluorescence intensity reflected the ATP concentration (RLU value).

[0251] ROS accumulation: The DCFH-DA probe was incubated for 30 minutes, and the green fluorescence intensity (MFI) was detected by flow cytometry.

[0252] Cell proliferation: EdU labeling for 2 hours, followed by Click-iT reaction to detect the EdU positivity rate (%).

[0253] Experimental data

[0254] Table 4: Effects of the metabolic synergistic module on energy metabolism and antioxidant capacity

[0255] Group ATP level (RLU×10³) ROS Accumulation (MFI) EdU positivity rate (%) P-value (vs. of the invention group) This invention group 58.3±4.7 1320±185 73.5±3.2 - Comparative Example 14 24.6±3.1 3980±420 41.2±2.8 <0.01 Comparative Example 15 36.8±3.9 2870±310 53.7±3.5 <0.01 Comparative Example 16 31.4±2.8 3520±380 47.6±3.1 <0.01 Comparative Example 17 29.5±3.3 4210±450 43.9±2.9 <0.01 Comparative Example 18 33.2±3.6 3680±410 51.3±3.4 <0.01 Comparative Example 19 45.1±4.1 2140±270 62.4±3.0 <0.05

[0256] Data Description

[0257] ATP levels: CellTiter-Glo was used to detect chemiluminescence intensity (n=5, mean ± SD), and RLU values ​​were positively correlated with ATP concentration.

[0258] ROS accumulation: The mean fluorescence intensity (MFI) was detected by flow cytometry using the DCFH-DA probe. A higher value indicates more severe oxidative stress.

[0259] EdU positivity rate: the proportion of actively proliferating cells as determined by flow cytometry (n=5, mean ± SD).

[0260] Statistical analysis: After one-way ANOVA, Dunnett's multiple comparison test showed that P < 0.05 and P < 0.01.

[0261] Experiment Summary

[0262] This experiment, through the systematic replacement or deletion of key components (biotin, tetramethylenediamine, and a mixture of vitamins) in a metabolic module, revealed the synergistic regulatory mechanism of stem cell energy metabolism and redox balance. Data showed that biotin, as a cofactor for carboxylases, directly participates in the rate-limiting steps of glycolysis (pyruvate carboxylase) and fatty acid synthesis (acetyl-CoA carboxylase); its deficiency (Comparative Example 14) led to a 57.8% decrease in ATP levels. While thiamine (Comparative Example 15) could partially compensate for the TCA cycle, its inability to support lipid metabolism resulted in a 45.3% reduction in ROS scavenging efficiency. Tetramethylenediamine's metal ion chelating function stabilizes Fe²⁺. + / Mn² + The activity of antioxidant enzymes (such as SOD and catalase) was inhibited, thus suppressing the burst of hydroxyl radicals induced by the Fenton reaction (comparative example 16 showed a cumulative increase of 166.7% in ROS), while EDTA (comparative example 17) excessively chelated essential trace elements (such as Zn²⁺). + Instead, it exacerbated metabolic disorders (ATP levels decreased by 49.4%).

[0263] The synergistic antioxidant network of the vitamin complex (vitamins C / E / B12) forms a multi-layered defense system by regenerating glutathione (GSH) and scavenging lipid peroxides. While N-acetylcysteine ​​(Comparative Example 19) can directly neutralize ROS with thiol groups (ROS reduction of 37.9%), its single effect cannot cover the protective function of the vitamin complex on mitochondrial membrane potential (ΔΨm), resulting in a still 15.1% decrease in proliferation rate. The innovation of this metabolic module lies in its multi-target coverage: biotin drives carbon flow redistribution (glycolysis → fatty acid synthesis), tetramethylenediamine optimizes metalloenzyme activity, and the vitamin complex constructs an antioxidant buffer; the three maintain metabolic homeostasis through spatiotemporal complementarity. For example, Comparative Example 18 (without vitamins) lacked a fat-soluble antioxidant (vitamin E), and ROS leaking from the mitochondrial electron transport chain could not be cleared in time, leading to DNA damage and proliferation inhibition (EdU positivity rate decreased by 30.3%).

[0264] In summary, the metabolic synergistic design of this invention breaks through the limitations of traditional culture systems that rely solely on glucose / glutamine for energy. Through nutrient metabolism coupling (biotin-carboxylation reaction), ion homeostasis regulation (tetramethylenediamine-metal balance), and redox network optimization (vitamin-free radical scavenging), a precise balance between energy supply and oxidative stress is achieved.

[0265] Please see Figure 2 The present invention also provides a culture medium system for maintaining and directionally inducing embryonic stem cell pluripotency, comprising:

[0266] The culture medium module is responsible for providing the necessary nutrients for maintaining and inducing embryonic stem cells. This typically includes, but is not limited to, amino acids, vitamins, minerals, and essential growth factors (such as LIF and bFGF). Its specific composition needs to be adjusted according to the target cell type in the experiment. The composition of the culture medium will be formulated according to different culture requirements, such as pluripotency maintenance medium or directed induction medium.

[0267] The temperature control module ensures the culture environment remains stable within the range of 36–38°C through a precise temperature control system (such as a water bath heater or incubator). Precise temperature control is crucial for the proliferation and metabolic activities of embryonic stem cells. Temperatures that are too high or too low can lead to cell growth arrest or death; therefore, maintaining a stable temperature range is key to ensuring long-term healthy cell culture.

[0268] The gas regulation module precisely controls the oxygen and carbon dioxide ratio in the culture environment, ensuring that the cell culture environment matches the in vivo microenvironment. The carbon dioxide concentration is typically controlled at 4%–6% to maintain the pH of the culture medium within the optimal range (7.2–7.4). The oxygen concentration is typically set at 20%–21% to simulate the normal metabolic needs of cells under physiological conditions.

[0269] The pH control module monitors and adjusts the pH of the culture medium to ensure it remains within the optimal range of 7.2–7.4. This is typically achieved using a pH sensor and an automated acid or alkali addition system. Embryonic stem cells are prone to metabolic problems or differentiation in pH-unstable environments; pH regulation can effectively prevent cellular stress responses and maintain their proliferative state.

[0270] The filtration and sterilization module is used to sterilize the base mixture using a 0.22μm filter membrane, effectively removing microorganisms, bacteria, and other impurities from the liquid to ensure the sterility and high purity of the final mixture. This module employs a high-efficiency filter membrane, which, through physical isolation, can precisely filter out microorganisms larger than 0.22μm in diameter while avoiding any potential sources of contamination, thus providing reliable and clean samples for subsequent experiments.

[0271] The storage module, used to store the culture medium, is typically stored in the dark at a low temperature of 2–8°C. Low-temperature storage helps prevent the degradation of growth factors and other nutrients, while also avoiding contamination of the culture medium. The storage module should have temperature control and UV protection functions to ensure the stability of the culture medium during long-term storage.

[0272] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A culture medium for maintaining and directionally inducing pluripotency of embryonic stem cells, characterized in that, The culture medium comprises the following components in parts by weight: Human insulin: 8–12 parts; Tetramethylene diamine: 0.5–0.8 parts; Luteinizing hormone: 0.2–0.5 parts; Vitamin mixture: 1-2 parts; Bovine serum albumin: 2-4 parts; Sodium bicarbonate: 0.5–1.0 parts; Antibiotic mixture: 0.2–0.4 parts; Biotin: 1-2 parts; Bone morphogenetic protein: 0.5–1.0 parts; Magnesium ethylenediaminetetraacetate: 0.1–0.2 parts; Oligopeptide-1: 2-4 parts; Interleukin-6: 2-4 parts; Phospholipid-encapsulated basic fibroblast growth factor: 1-2 parts; PD0325901: 0.05–0.1 parts; Dickkopf-1: 0.01–0.05 parts; Polypeptide-modified sodium hyaluronate: 0.1–0.3 parts; The human insulin is recombinant human insulin; the tetramethylene diamine is of cell culture grade purity with a purity >99%; the luteinizing hormone is selected from naturally extracted or synthetic progesterone hormones; the vitamin mixture includes vitamin A, vitamin C, vitamin E, and B vitamins, with vitamin A: 1-2, vitamin C: 10-20, vitamin E: 0.5-1, and B vitamins: 1-2; the bovine serum albumin is low endotoxin grade free fatty acid albumin; and the sodium bicarbonate is analytical grade or cell culture grade. The antibiotic mixture includes penicillin and streptomycin, with penicillin at 100-200 U / mL and streptomycin at 100-200 μg / mL. The biotin is water-soluble D-biotin powder, the bone morphogenetic protein is bone morphogenetic protein 4, the magnesium ethylenediaminetetraacetate is a complexing solution prepared with EDTA and MgCl2 at a molar ratio of 1:1, the oligopeptide-1 is recombinant human oligopeptide-1 from the epidermal growth factor family, and the interleukin-6 is recombinant human interleukin-6 from the interleukin cytokine family. The phospholipid-encapsulated basic fibroblast growth factor is a liposome, and the liposome particle size is controlled between 100 and 300 nm; the vitamin mixture is an emulsified vitamin mixture with a particle size ≤ 100 nm.

2. A method for preparing a culture medium for maintaining and directed inducing embryonic stem cell pluripotency, applied to the culture medium for maintaining and directed inducing embryonic stem cell pluripotency as described in claim 1, characterized in that, Includes the following steps: S1. Take deionized water and place it in a constant temperature stirring device. Add human insulin, tetramethylenediamine, dissolved progesterone, emulsified vitamin mixture, bovine serum albumin and biotin in sequence and stir continuously to form a preliminary mixture. Add sodium bicarbonate to the preliminary mixture to adjust the pH to 7.2-7.4, then add antibiotic mixture and continue stirring to obtain basic mixture. S2. The basic mixture is sterilized by filtration through a 0.22μm filter membrane to obtain a sterile basic solution; S3. Under aseptic conditions, bone morphogenetic protein, magnesium ethylenediaminetetraacetate, oligopeptide-1 and interleukin-6 are dissolved in sterile deionized water to prepare separate premixes. The separate premixes are added to the sterile base solution and stirred to obtain a composite mixture. S4. Add phospholipid-encapsulated basic fibroblast growth factor liposomes to the composite mixture and stir to mix. S5. Dissolve PD0325901 in anhydrous ethanol, dilute with sterile deionized water and add to the composite mixture. Then add recombinant Dickkopf-1 protein and peptide-modified sodium hyaluronate, stir to obtain a homogeneous mixture, seal and dispense into containers to obtain the culture medium.

3. The method for preparing the culture medium for maintaining and directionally inducing embryonic stem cell pluripotency according to claim 2, characterized in that, In step S1, 1000 mL to 1500 mL of deionized water is placed in a constant temperature stirring device. Human insulin, tetramethylene diamine, dissolved progesterone, emulsified vitamin mixture, bovine serum albumin, and biotin are added sequentially. The mixture is stirred continuously at 37°C and 300 to 600 rpm for 20 to 40 minutes to form a preliminary mixture. Sodium bicarbonate is added to the preliminary mixture to adjust the pH to 7.2 to 7.

4. Then, the antibiotic mixture is added, and the mixture is stirred for another 10 to 30 minutes to obtain the basic mixture.

4. The method for preparing the culture medium for maintaining and directionally inducing embryonic stem cell pluripotency according to claim 2, characterized in that, In step S3, the luteinizing hormone is pre-dissolved in anhydrous ethanol with a volume fraction of 95% at a mass ratio of 1:5 to 1:10, and then 0.1% to 0.5% of polysorbate-80 is added as an emulsifier. The mixture is stirred for 10 to 20 minutes at 40 to 50°C and 200 to 400 rpm to obtain the dissolved luteinizing hormone. The vitamin A and vitamin E in the vitamin mixture are mixed at a mass ratio of 1:1 to 1:2, dissolved in anhydrous ethanol with a volume fraction of 90% to 95%, and homogenized with lecithin at a mass ratio of 1:2 to 1:5 at 50 to 60°C and 500 to 800 rpm for 15 to 30 minutes. The emulsified vitamin mixture has a particle size ≤100 nm. The dissolved progesterone and emulsified vitamin mixture is added simultaneously with deionized water in step S1.

5. The method for preparing the culture medium for maintaining and directionally inducing embryonic stem cell pluripotency according to claim 2, characterized in that, In step S3, bone morphogenetic protein, magnesium ethylenediaminetetraacetate, oligopeptide-1 and interleukin-6 are dissolved in 10-20 mL of sterile deionized water to prepare individual premixes. The individual premixes are added to the sterile base solution and stirred at 36-38°C and 300 rpm for 15-45 minutes to obtain a composite mixture.

6. The method for preparing the culture medium for maintaining and directionally inducing embryonic stem cell pluripotency according to claim 2, characterized in that, In step S4, phospholipid-encapsulated basic fibroblast growth factor liposomes are added to the composite mixture. The particle size of the liposomes is controlled at 100-300 nm, the stirring rate is maintained at 200-400 rpm, and the mixture is continued for 15-30 minutes.

7. The method for preparing a culture medium for maintaining and directionally inducing embryonic stem cell pluripotency according to claim 2, characterized in that, In step S5, PD0325901 is dissolved in 1-2 mL of anhydrous ethanol with a content of 92-95% at a mass ratio of 1:50 to 1:

100. After dilution with 5-10 mL of sterile deionized water, it is slowly added dropwise to the composite mixture to control the final concentration within the range of 0.05-0.1 parts, and the system temperature is maintained at 35-37°C.

8. The application of the culture medium for maintaining and directing the pluripotency of embryonic stem cells as described in claim 1 in the differentiation and reprogramming of embryonic stem cells.

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