Healthy individual source autologous tumor stem cell line building method

Abstract

The invention discloses a healthy individual-derived autologous tumor stem cell line establishment method, which is characterized by comprising the following two stages: a first stage, inducing healthy human-derived somatic cells into hiPSC; in the second stage, the hiPSC is induced to be converted into the tumor stem cells, specifically, firstly, a tumor-derived conditioned culture medium is prepared, then under the synergistic effect of the tumor-derived conditioned culture medium and an induction adjuvant, the hiPSC is induced to be converted into the tumor stem cells, and the healthy individual-derived tumor stem cells are obtained after culture. According to the invention, the somatic cell-derived iPSC of healthy people can be induced into tumor stem cells, and the method can be well applied to cancer prevention, drug screening and health management.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to a method for technically transforming healthy human cells into tumor stem cells. Background Technology

[0002] In 2022, China reported approximately 4.82 million new cases of cancer and 2.57 million deaths. Even today, cancer remains a major public health problem in China, seriously threatening the survival and health of its residents.

[0003] Cancer stem cells (CSCs), also known as tumor-initiating cells (TICs), are a subpopulation of cells with self-renewal, unlimited proliferative potential, multidrug resistance, and the ability to drive tumor recurrence and metastasis. Their origins include the malignant transformation of normal stem cells or the dedifferentiation of differentiated tumor cells, making them the root cause of cancer occurrence, recurrence, and metastasis. Effective prevention methods can significantly reduce cancer incidence and mortality. Cancer stem cell inhibition is a crucial step in cancer prevention and has significant implications for human health. However, the number of cancer stem cells within human tumors is extremely small, making capture difficult, culture costly, and time-consuming, hindering large-scale in vitro culture. Establishing a cancer stem cell drug screening and evaluation platform is also technically challenging, greatly limiting drug research targeting cancer stem cells. Furthermore, cancer stem cells cannot be obtained from healthy individuals; there are currently no reports of cancer stem cells derived from healthy populations. Therefore, how to innovatively establish a cancer stem cell model derived from healthy individuals for application in drug screening and health management is an urgent problem to be solved.

[0004] In 2006, scientist Yamanaka used four pluripotency factors—Oct4, Sox2, Klf4, and c-Myc—to reprogram mouse fibroblasts into induced pluripotent stem cells (iPSCs), similar to mouse embryonic stem cells, possessing the potential for unlimited proliferation and differentiation. Subsequently, multiple research groups reported the successful acquisition of human iPSCs. The emergence of iPSCs has profound significance, serving as a powerful tool for researching disease models, drug screening, and cell therapy. iPSCs and tumor stem cells share many commonalities: 1. iPSCs and CSCs are highly similar at the molecular level, including sharing multiple antigen expression profiles and key signaling pathways; both iPSCs and tumor stem cells express tumor stem cell markers; 2. Both iPSCs and CSCs possess continuous self-renewal capabilities. CSCs can proliferate indefinitely and differentiate into heterogeneous tumor cell populations, while iPSCs, as pluripotent stem cells, also possess the potential for self-renewal and differentiation into various cell types.

[0005] Based on the above description, this invention aims to transform iPSCs derived from healthy individuals into malignant cell characteristics through key technological modifications, thereby converting them into tumor stem cells. Summary of the Invention

[0006] Based on existing technology, this invention aims to provide a method for establishing autologous tumor stem cell lines from healthy individuals, which can induce iPSCs from somatic cells of healthy people into tumor stem cells, and can be well applied to cancer prevention, drug screening and health management.

[0007] The technical terms used in this invention are as follows: peripheral blood mononuclear cells: PBMC; induced pluripotent stem cells: iPSC; human induced pluripotent stem cells: hiPSC.

[0008] To achieve the purpose of this invention, the present invention provides a method for establishing autologous tumor stem cell lines from healthy individuals, characterized by comprising the following two stages: The first stage involves inducing somatic cells from healthy individuals into hiPSCs: Somatic cells from healthy individuals are extracted and reprogrammed using plasmids containing four reprogramming factors, OCT4, c-MYC, KLF-4, and SOX2. The cells are then cultured in iPSC medium to obtain hiPSCs. The generated hiPSCs are identified by alkaline phosphatase and immunofluorescence staining for pluripotency markers to ensure that the correct hiPSCs are obtained. The second stage involves inducing hiPSCs to transform into tumor stem cells. First, tumor-derived conditional culture medium is prepared: human tumor cells are obtained and cultured in high-glucose DMEM medium containing 10% fetal bovine serum until the confluence reaches 70-80%. Then, the medium is completely replaced with high-glucose DMEM medium containing 5% FBS and 1% Anti-Anti. After culturing for 48 hours, the medium is collected, centrifuged at 1000 g for 10 minutes, and the supernatant is collected. After filtration through a 0.22 μm filter membrane, the tumor-derived conditional culture medium is obtained. Then, under the synergistic effect of tumor-derived conditioned medium and inducing adjuvants, hiPSCs were induced to transform into tumor stem cells. The inducing adjuvants included, but were not limited to, CHIR, PD0325901, AZD3244, hLIF, and FGF2. After several weeks of induction culture, the cells were digested with trypsin and identified by flow cytometry. Cells expressing tumor stem cell markers were identified as tumor stem cells derived from healthy individuals.

[0009] The method provided by this invention can induce iPSCs derived from peripheral blood somatic cells of healthy individuals into tumor stem cells and express tumor stem cell markers.

[0010] In the second stage of the method of the present invention, hiPSCs are induced to transform into tumor stem cells. The inducing adjuvant used is an immunological reagent, such as an antibody, peptide or small molecule, to enhance or improve the transformation of iPSCs into tumor stem cells.

[0011] Furthermore, in the first stage of the method, the somatic cells derived from healthy individuals are: peripheral blood mononuclear cells (PBMCs) derived from the peripheral blood of healthy individuals, or cells extracted from the skin, muscle, fat, bone marrow, organs, hair, urine, or any combination thereof from healthy individuals.

[0012] Given the similarity between iPSCs derived from peripheral blood PBMCs of healthy individuals and iPSCs derived from somatic cells of various tissues and organs, iPSCs can be generated using one's own tissues (e.g., skin, muscle, fat, bone marrow, organs, hair, blood, urine, or combinations of tissues) to produce tumor stem cells.

[0013] Furthermore, the first stage of the method involves preparing hiPSCs using a non-integrative plasmid containing four reprogramming factors: OCT4, c-MYC, KLF-4, and SOX2, as well as possible shRNAp53.

[0014] Furthermore, in the first stage of the method, human iPSCs are prepared by reprogramming cells using plasmids containing four reprogramming factors OCT4, c-MYC, KLF-4 and SOX2, as well as plasmids containing any of the following pluripotency genes: Nanog, Lin28, L-MYC, SALL4, and TBX3.

[0015] Furthermore, in the first stage of the method, non-integrating plasmids are used as vectors for reprogramming factors, or lentiviruses, Sendai viruses, PiggyBac, and Sleepy Beauty integrating plasmids are used as vectors for reprogramming factors to induce somatic cells to transform into iPSCs through genome reprogramming.

[0016] Furthermore, in the first stage of the method, iPSCs are prepared by electroporation of plasmids including OCT4, c-MYC, KLF-4 and SOX2 into PBMC cells for reprogramming.

[0017] Furthermore, the second stage of the method, the preparation method of tumor-derived conditional culture medium, includes the following steps: (1) Obtain tumor cells, inoculate them in high glucose DMEM medium containing 10% fetal bovine serum, and incubate them in a constant temperature incubator at 37°C and 5% CO2. Replace the medium with fresh medium the next day to remove the residual cryopreservation solution. (2) After the cells have stabilized, passage them once and continue to expand the culture until the cell confluence reaches 70%-80%; (3) Remove the original culture medium, gently wash the cells 1-2 times with DPBS buffer, and then replace it with high-glucose DMEM medium containing 5% FBS and 1% Anti-Anti. Incubate at 37°C and 5% CO2 for 48 hours. (4) After 48 hours, collect the culture medium, centrifuge at 1000 g for 10 minutes and collect the supernatant. Then filter it with a 0.22 μm filter membrane, take a small amount of the filtrate and add it to a six-well plate and let it stand overnight for culture. If no surviving cells remain in the filtrate, then qualified tumor-derived conditional culture medium is obtained.

[0018] Furthermore, the second stage of the method, the process of inducing hiPSCs to transform into tumor stem cells, includes the following steps: (1). HiPSCs were seeded into six-well plates coated with VTN (Vitronectin), and iPSC medium was used. The cells were then cultured in a 37°C, 5% CO2 incubator. (2). After 24 hours, the induction medium was changed to: iPSC medium and tumor-derived conditional medium were mixed at a ratio of 1:2 to 1:1, and 10-100 μM AZD3244, 1-10 μM CDIR99021, 1000-10000 U / mL human leukemia inhibitory factor (hLIF), and 5-50 ng / mL FGF2 were added. The mixture was then placed in a 37℃, 5% CO2 incubator for culture. (3) Replace half of the culture medium daily (half the volume of medium), and observe the changes in cell morphology and proliferation under an inverted microscope, and record the cell status; (4) When the cell confluence reaches 70%-80%, digest the cells with iPS mild digestion solution and passage them into new VTN-coated culture dishes at a ratio of 1:5-1:8. Continue to culture under the same induction conditions. When the iPSCs morphology shows tightly packed round clumps, the clumps become larger and thicker and there are irregular single cells distributed around the clumps, trypsin is used to induce cell digestion and the cell line is identified by flow cytometry.

[0019] The iPSC medium of this invention is 2i / LIF. Given the similarity of the components of various media, it can also be StemBasic03, Essential 8, Stem Flex, mTESR1, etc.

[0020] Furthermore, in the second stage of the method, when preparing the tumor-derived conditional culture medium, the tumor cells can be selected from any type of cancer, including solid tumors and hematological malignancies. The tumor cell-derived conditional culture medium and inducing adjuvant are used to induce the transformation of human iPSCs into tumor stem cells, thereby establishing tumor stem cells derived from iPSCs of healthy individuals.

[0021] The method of this invention is applicable to a wide variety of cancer types, encompassing all cancer categories. Based on the similarity between iPSCs and tumor stem cells, it is possible to establish tumor stem cells derived from iPSCs in healthy individuals for solid tumors (such as breast cancer, lung cancer, skin cancer, glioblastoma, head and neck cancer, thyroid cancer, pancreatic cancer, liver cancer, colorectal cancer, kidney cancer, stomach cancer, sarcoma, ovarian cancer, bladder cancer, prostate cancer, esophageal cancer, endometrial cancer, and cervical cancer) as well as for hematological malignancies (including Hodgkin's lymphoma, non-Hodgkin's lymphoma, multiple myeloma, spinal proliferative disease, and leukemia).

[0022] In a preferred embodiment, in the second stage of the method, when preparing the tumor-derived conditional culture medium, the tumor cells are derived from the hepatocellular carcinoma cell line Huh7, thereby obtaining the conditional culture medium derived from the hepatocellular carcinoma cell line Huh7; using the conditional culture medium derived from the hepatocellular carcinoma cell line Huh7 and an inducing adjuvant to induce the transformation of human iPSCs into hepatocellular carcinoma stem cells, and culturing to obtain cells that are positive for expressing markers CD24, CD44, and EpCAM, which are hepatocellular carcinoma stem cells derived from healthy individuals.

[0023] As an alternative to the above preferred embodiment, when preparing the tumor-derived conditional culture medium, the following human liver cancer cell lines can also be used as the source of tumor cells: Hep3B, HepG2, HCCLM3, MHCC97H, HAK-5, SMCC-7721, BEL-7402, PLC / PRF / 5, SMMC-7221, and Bel-7402 instead of Huh7.

[0024] Beneficial effects of the invention

[0025] This invention provides a method for establishing autologous tumor stem cell lines from healthy individuals, which can induce iPSCs derived from somatic cells in healthy individuals into tumor stem cells. The induction cycle is short, efficient, and low-cost. Furthermore, the tumor stem cell model derived from healthy individuals established by this invention can be well applied to cancer prevention, drug screening, and health management. Attached Figure Description

[0026] Figure 1 The iPSC morphology of human PBMCs cultured in Example 1; Figure 2 Immunofluorescence staining was used to identify human PBMC-derived iPSCs cultured in Example 1; Figure 3 The morphology on day 30 induced by iPSC-Huh7-1 in Example 2; Figure 4 This is a negative control chromatogram from the flow cytometry analysis in Example 2; Figure 4 Explanation of the markings in the text: Figure 4-1Gate the scattered light; Figure 4-2 It belongs to the unicellular phylum; Figure 4-3 A gate is set for FITC+PE dual-color negative; Figure 4-4 A gate is set for FITC+APC two-color negative; Figure 4-5 A gate is set for PE+APC dual-color negative; Figure 5 The flow cytometry analysis diagram of iPSC-Huh7-1 in Example 2; Figure 5 Explanation of the markings in the text: Figure 5-1 Gate the scattered light; Figure 5-2 It belongs to the unicellular phylum; Figure 5-3 The co-positive rate was 40.85% for both CD24 and CD44. Figure 5-4 The co-positive rate was 14.71% for CD24 and EpCAM. Figure 5-5 The co-positive rate was 12.16% for CD44 and EpCAM. Detailed Implementation

[0027] To better understand the invention and more clearly describe the various steps and operation schemes of the invention, the technical solution of the invention will be described in detail below with reference to the accompanying drawings and embodiments.

[0028] Example 1: Induction of somatic cells from healthy individuals into iPSCs and identification of pluripotency

[0029] This example illustrates the induction of iPSCs from peripheral blood mononuclear cells (PBMCs) derived from healthy individuals. The specific procedures are as follows: (a) Methods for inducing peripheral blood mononuclear cells (PBMCs) into iPSCs include the following procedures: 1. Peripheral blood mononuclear cells (PBMCs) were extracted from peripheral blood of healthy individuals, and the PBMCs were cultured. Sterilized PBS solution and electrolysis solution were prepared, and the electrolysis cup was disinfected with alcohol and then air-dried in a laminar flow hood and sterilized with ultraviolet light for 30 min. 2. Start the cell electroporator, set the PBMC electroporation parameters, and count the PBMC cells every 10... 6 Add 100 μL of electrolysis solution and 5 μg of plasmid DNA encoding the reprogrammed genes OCT4, c-MYC, KLF-4 and SOX2 to each cell, and mix carefully with a pipette tip to avoid generating air bubbles. 3. After mixing, quickly add the PBMC and plasmid suspension into the electroporation cup, avoiding the generation of air bubbles, and place it into the electroporation apparatus for electroporation. 4. Quickly resuspend the transfected cell suspension in an appropriate amount of N2B27+FGF2 medium, and transfer the mixture to the appropriate culture dish. Incubate at 37°C in a 5%-7.5% CO2 cell incubator. 5. On day 7 after electroporation, cell morphology changed, and clonal patterns appeared. The culture medium was then changed to 2i / h LIF-iPSC medium. Around day 14 after electroporation, clones were basically formed, with morphology similar to mouse ES cells. The medium was changed again, and the cells were cultured for 21 days. Clones were then picked, passaged, expanded, and cultured until the iPSC morphology was as shown in the image. Figure 1 As shown.

[0030] (II) Identification of iPSC pluripotency 1. Dilute the primary and secondary antibodies against pluripotency factor to appropriate concentrations using blocking buffer (the secondary antibody should be diluted in the dark); dilute DAPI with PBS solution (DAPI should be diluted in the dark) to appropriate concentrations (dilution ratio 1:10,000); human iPS cells should be grown in 24-well plates until 50%-60% coverage is achieved. 2. Use a vacuum filter pump and pipette tip to aspirate the old culture medium, add 1 mL of DPBS buffer to a 24-well cell culture dish, gently shake to infiltrate the cells, aspirate the DPBS buffer, and repeat once; 3. Add 1 mL of 4% paraformaldehyde along the edge of the petri dish and fix at room temperature in the dark for 30 min; 4. Add 1 mL of DPBS buffer to infiltrate the cells for 5 min, discard the waste liquid, and repeat three times; 5. Add 0.2% Triton and treat for 15 min (for nucleostained staining).

[0031] 6. Add 1 mL of blocking buffer and incubate at room temperature for 1 h; add 500 μL of primary antibody and incubate overnight at 4°C; 7. Add 1 mL of DPBS buffer and soak for 5 min, then discard the waste liquid. Repeat three times. 8. Add 500 μL of the diluted secondary antibody under light-protected conditions and incubate at room temperature in the dark for 30 min; 9. Infiltrate cells with 1 mL of DPBS buffer for 5 min under light-protected conditions, discard the waste solution, and repeat three times; 10. Add 500 μL of diluted DAPI under light-protected conditions and incubate at room temperature for 1-2 min; add 1 mL of DPBS buffer to infiltrate the cells for 5 min under light-protected conditions, aspirate the waste liquid, and repeat three times; then add an appropriate amount of DPBS and take pictures and record them under an inverted fluorescence microscope under light-protected and room temperature conditions.

[0032] The immunofluorescence staining identification results of human PBMC-derived iPSCs cultured in Example 1 of this invention are as follows: Figure 2 As shown: the iPSCs obtained in this invention express pluripotency markers such as OCT4, KLF4, SOX2, and NANOG, which meet the standards for pluripotent stem cells.

[0033] Example 2: Establishing a standardized iPSC to CSC induction differentiation scheme (a) Preparation of tumor-derived conditioned media This embodiment will specifically illustrate the preparation of conditional culture medium derived from the human hepatocellular carcinoma cell line Huh-7.

[0034] 1. Human hepatocellular carcinoma cells Huh-7 were revived from liquid nitrogen and inoculated into high-glucose DMEM medium containing 10% fetal bovine serum (High-DMEM + 10% FBS + 1% MEM NEAA (100×) + 1% L-Ala-Gln (100×) + 1% Sodium Pyruvate (100 mM) + 1% Anti-Anti). The medium was then incubated at 37°C in a 5% CO2 incubator. The medium was replaced with fresh medium the next day to remove any residual cryopreservation solution. 2. Once the cells have stabilized, passage them once; continue the culture until the cell confluence reaches 70%-80%.

[0035] 3. Remove the original culture medium, gently wash the cells 1-2 times with DPBS buffer, and then replace it with basal culture medium containing 5% FBS and 1% Anti-Anti (High-DMEM + 5% FBS + 1% Anti-Anti); place it in a 37℃, 5% CO2 incubator and continue to culture for 48 hours.

[0036] 4. After 48 hours, the culture supernatant was collected and centrifuged at 1,000×g for 10 minutes at room temperature to remove cell debris; then filtered through a 0.22 μm filter membrane to obtain sterile tumor-derived conditional medium (CM), specifically sterile huh7-derived conditional medium.

[0037] 5. Add 3 mL of the filtered CM solution to a six-well plate and incubate overnight. Only after confirming that there are no surviving cells remaining can it be used for subsequent experiments.

[0038] (ii) Establish a standardized iPSC to CSC induction differentiation scheme 1. Quantity 5 * 10 4 hiPSCs were seeded in six-well plates coated with VTN (Vitronectin), and cultured in 2i / h LIF-iPSC medium at 37°C in a 5% CO2 incubator. The induced experimental group was named hiPSC-HUH-7-1. 2. Replace the induction medium after 24 hours: On the basis of mixing 2i / hLIF-iPSC medium and Huh7-derived conditional medium at a ratio of 1:2 to 1:1, add 10-100 μM AZD3244, 1-10 μM CDIR99021, 1-10 μMPD0325901, 1000-10000 U / mL human leukemia inhibitory factor (hLIF), and 5-50 ng / mL FGF2; 3. Replace half of the culture medium daily (half the volume of medium) to maintain a stable factor concentration; observe cell morphology changes and proliferation under an inverted microscope daily and record cell status; the entire induction process lasts for one month; 4. When the cell confluence reaches 70%-80%, digest the cells using iPS mild digestion solution and passage them into new VTN-coated culture dishes at a ratio of 1:5-1:8. Continue culturing under the same induction conditions. On day 30 of induction, iPSCs begin to appear, exhibiting enlarged and thickened clumps with a tightly packed, round shape, and irregularly distributed single cells around the clumps. The morphology of iPSCs on day 30 of induction into liver cancer stem cells is as follows. Figure 3 .

[0039] (III) Flow Cytometry Detection 1. On day 30 of culture, the old culture medium in the culture dish of hiPSC-HUH-7-1 induction group and hiPSCs in D0 control group was discarded, and 2 mL of pre-warmed DPBS buffer was added to gently wash twice to remove residual metabolites, etc. 2. Add 1 mL of iPS mild digestion solution, observe the cell state under a microscope, and perform digestion in multiple steps. Add double the volume of Stemfit basic O3 medium containing 10% FBS to terminate the digestion. 3. Collect all liquid into a 15 mL centrifuge tube, rinse the culture dish once with 1 mL DPBS buffer, and transfer it into a 15 mL centrifuge tube; 4. Centrifuge the obtained cell suspension at 300×g at room temperature for 5 min, and discard the supernatant completely; 5. Resuspend the cells in 1 mL of DPBS buffer, use a 1 mL pipette with a 200 µL pipette tip to repeatedly pipette 10 times, and pass the cells through a 40 µm cell sieve to remove any remaining clumps. 6. Mix 10 µL of cell suspension with 10 µL of AO / PI staining solution at a 1:1 ratio, and use an automated cell counter to determine cell viability and total cell count; 7. Calculate the required volume based on the counting results, and take 5 × 10⁻⁶. 5Transfer the cells to a new 1.5 mL EP tube; centrifuge at 300×g at room temperature for 5 min, discard the supernatant, and remove any remaining liquid from the tube wall with a 200 µL pipette to obtain a tight cell pellet, thus avoiding antibody dilution errors; 8. Take two EP tubes and label them "Experimental Group" and "Negative Control" respectively. Experimental group: Cells were resuspended in 50 μL of flow cytometry staining buffer, followed by the addition of antibody; Negative control: Resuspend directly in 50 μL of flow cytometry staining buffer, without adding antibody subsequently; 9. Following the recommended amounts in the instructions, add the three antibodies CD24, CD44, and EpCAM to the same EP tube, and bring the volume to 50 μL with flow cytometry staining buffer. Mix gently. The antibody addition amounts are: CD24-FITC 5 µL / test, CD44-PE 0.125 µg / test, and EpCAM-APC 0.125 µg / test. 10. Staining the experimental group: Add 50 μL of antibody mixture to 50 μL of cell suspension in the experimental group, for a total volume of 100 μL, and mix gently; do not add antibody to the negative control tube; immediately transfer the experimental group and the negative control group to a 4°C metal bath or wet ice box and incubate in the dark for 30 min, gently tapping the tube wall once every 10 min to prevent cell sedimentation. 11. Add 500 µL of pre-cooled staining buffer to each tube, gently pipette three times, centrifuge at 300×g for 5 min, and discard the supernatant completely; 12. Repeat step 11; 13. Resuspend the precipitate in 500 μL of flow cytometry staining buffer and pipette until it reaches a single-cell state; 14. Run the negative control first: set the FSC / SSC voltage and gate region; then run the experimental group: collect ≥10,000-50,000 target cell events; 15. Data Recording: Export the flow cytometry data file (.fcs file). Subsequent flow cytometry plotting software will be used to analyze the expression ratios of CD24, CD44, and EpCAM. The flow cytometry analysis results are shown below. Figure 4 , Figure 5 .

[0040] Figure 4 A negative control figure for flow cytometry analysis ( Figure 4-1 Gate the scattered light; Figure 4-2 It belongs to the unicellular phylum; Figure 4-3 A gate is set for FITC+PE dual-color negative; Figure 4-4 A gate is set for FITC+APC two-color negative; Figure 4-5 (Set up a gate for PE+APC dual-color negative). Figure 5For iPSC-Huh7-1 flow cytometry analysis ( Figure 5-1 Gate the scattered light; Figure 5-2 It belongs to the unicellular phylum; Figure 5-3 The co-positive rate was 40.85% for both CD24 and CD44. Figure 5-4 The co-positive rate was 14.71% for CD24 and EpCAM. Figure 5-5 The co-positive rate was 12.16% for CD44 and EpCAM.

[0041] iPSCs and liver cancer stem cells co-express CD24 and CD44. EpCAM antibody is not expressed in iPSCs, therefore EpCAM can be considered a specific marker for liver cancer stem cells. CD24, CD44, and EpCAM are surface markers of liver cancer stem cells.

[0042] like Figure 4 , Figure 5 As shown, the control group iPSCs expressed CD24 and CD44 markers, but no hepatocellular carcinoma stem cells expressed EpCAM. The experimental group iPSC-Huh7-1 expressed CD24 and CD44, common markers of iPSCs and hepatocellular carcinoma stem cells, and also expressed EpCAM, the positive rate of CD24-CD44-EpCAM, which was 20.88%, indicating the generation of hepatocellular carcinoma stem cells derived from iPSCs.

[0043] The embodiments of this invention are merely illustrative of the technical solutions of this invention and are not intended to limit the scope of protection of this invention. Those skilled in the art can adjust and optimize the implementation methods based on the guidance of this invention without departing from the core concept of the invention. Technical solutions obtained by replacing the same or equivalent technical means without creative effort are all within the scope of protection of the claims of this invention.

Claims

1. A method for establishing autologous tumor stem cell lines from healthy individuals, characterized in that, It includes the following two stages: The first stage involves inducing somatic cells from healthy individuals into hiPSCs: Somatic cells from healthy individuals are extracted and reprogrammed using plasmids containing four reprogramming factors, OCT4, c-MYC, KLF-4, and SOX2. The cells are then cultured in iPSC medium to obtain hiPSCs. The generated hiPSCs are identified by alkaline phosphatase and immunofluorescence staining for pluripotency markers to ensure that the correct hiPSCs are obtained. The second stage involves inducing hiPSCs to transform into tumor stem cells. First, tumor-derived conditional culture medium is prepared: human tumor cells are obtained and cultured in high-glucose DMEM medium containing 10% fetal bovine serum until the confluence reaches 70-80%. Then, the medium is completely replaced with high-glucose DMEM medium containing 5% FBS and 1% Anti-Anti. After culturing for 48 hours, the medium is collected, centrifuged at 1000 g for 10 minutes, and the supernatant is collected. After filtration through a 0.22 μm filter membrane, the tumor-derived conditional culture medium is obtained. Then, under the synergistic effect of tumor-derived conditioned medium and inducing adjuvants, hiPSCs were induced to transform into tumor stem cells. The inducing adjuvants included, but were not limited to, CHIR, PD0325901, AZD3244, hLIF, and FGF2. After several weeks of induction culture, the cells were digested with trypsin and identified by flow cytometry. Cells expressing tumor stem cell markers were identified as tumor stem cells derived from healthy individuals.

2. The method according to claim 1, characterized in that, In the first stage, human iPSCs were prepared by reprogramming cells using plasmids containing four reprogramming factors: OCT4, c-MYC, KLF-4, and SOX2, as well as any of the following pluripotency genes: Nanog, Lin28, L-MYC, SALL4, and TBX3.

3. The method according to claim 1, characterized in that, In the first stage, the somatic cells derived from healthy individuals are: peripheral blood mononuclear cells derived from the peripheral blood of healthy individuals, or cells extracted from the skin, muscle, fat, bone marrow, organs, hair, urine, or any combination thereof of healthy individuals.

4. The method according to claim 1, characterized in that, In the first stage, non-integrating plasmids are used as vectors for reprogramming factors, or lentiviruses, Sendai viruses, PiggyBac, and Sleepy Beauty integrating plasmids are used as vectors for reprogramming factors to induce somatic cells to transform into iPSCs through genome reprogramming.

5. The method according to claim 1, characterized in that, The first stage involves the reprogramming of iPSCs by electroporating plasmids containing OCT4, c-MYC, KLF-4, and SOX2 into PBMC cells.

6. The method according to claim 1, characterized in that, In the second stage, when preparing the tumor-derived conditional culture medium, the tumor cells can be selected from any type of cancer, including solid tumors and hematological malignancies. The tumor cell-derived conditional culture medium and inducing adjuvant are used to induce the transformation of human iPSCs into tumor stem cells, thereby establishing tumor stem cells derived from iPSCs of healthy individuals.

7. The method according to claim 1, characterized in that, In the second stage, when preparing the tumor-derived conditional culture medium, the tumor cells are derived from the hepatocellular carcinoma cell line Huh7, thereby obtaining the conditional culture medium derived from the hepatocellular carcinoma cell line Huh7; using the conditional culture medium derived from the hepatocellular carcinoma cell line Huh7 and an inducing adjuvant to induce the transformation of human iPSCs into hepatocellular carcinoma stem cells, and culturing to obtain cells that are positive for the expression markers CD24, CD44, and EpCAM, which are hepatocellular carcinoma stem cells derived from healthy individuals.

8. The method according to claim 7, characterized in that, When preparing the conditional culture medium for tumor-derived cells, the following human liver cancer cell lines can also be used as sources of tumor cells: Hep3B, HepG2, HCCLM3, MHCC97H, HAK-5, SMCC-7721, BEL-7402, PLC / PRF / 5, SMMC-7221, and Bel-7402, instead of Huh7.

9. The method according to claim 1, characterized in that, In the second stage, the process of inducing hiPSCs to transform into tumor stem cells includes the following steps: (1) HiPSCs cells were seeded into VTN-coated six-well plates, and iPSC medium was used. The cells were then cultured in a 37°C, 5% CO2 incubator. (2) After 24 hours, the induction medium was changed to: iPSC medium and tumor-derived conditional medium were mixed at a ratio of 1:2 to 1:1, and 10-100 μM AZD3244, 1-10 μM CDIR99021, 1000-10000 U / mL hLIF, and 5-50 ng / mL FGF2 were added; and the medium was placed in a 37℃, 5% CO2 constant temperature incubator for incubation. (3) Replace half of the culture medium daily and observe cell morphological changes and proliferation under an inverted microscope, and record the cell status; (4) When the cell confluence reaches 70%-80%, digest the cells with iPS mild digestion solution and passage them into new VTN-coated culture dishes at a ratio of 1:5 to 1:

8. Continue to culture under the same induction conditions as in step (2). When the iPSCs morphology shows tightly packed round clumps, the clumps become larger and thicker and there are irregular single cells distributed around the clumps, digest the cells with trypsin to induce them and identify them by flow cytometry.

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