A method for eliminating residual pluripotent stem cells after pluripotent stem cell differentiation

Abstract

The present invention discloses a method for removing residual pluripotent stem cells after differentiation of pluripotent stem cells. The present invention develops a method for removing residual pluripotent stem cells that is harmless to cells, has a high cost-effectiveness, and can efficiently remove residual pluripotent stem cells. The method uses a balanced salt solution that does not contain calcium ions and magnesium ions to achieve efficient removal of residual pluripotent stem cells, reduce the risk of immature teratomas, and has important clinical application value.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and specifically relates to a method for removing residual pluripotent stem cells after pluripotent stem cell differentiation, and more specifically, relates to a method for removing residual pluripotent stem cells after pluripotent stem cell differentiation using a balanced salt buffer solution free of calcium ions and magnesium ions. Background Art

[0002] Tissue engineering and cell therapy based on pluripotent stem cells (PSCs) and their differentiated cells is a promising therapeutic approach that has been applied in clinical and preclinical studies of a variety of diseases, including diabetes, acute stroke, macular degeneration, ischemic cardiomyopathy, etc. However, PSCs and their derived cells and tissues may contain residual, undifferentiated PSCs, which may lead to the formation of immature teratomas after implantation into patients. Therefore, there is an urgent need in this field to develop a new method for removing residual undifferentiated PSCs that does not affect the differentiation process of PSCs, which is simple and effective, so as to achieve its safe clinical application.

[0003] Currently, various methods have been used in attempts to eliminate residual PSCs, such as genetic modification, small molecule treatment, and physical therapy. Among them, genetic modification is currently the most mainstream PSC elimination solution. This method achieves efficient in vitro clearance of iPSCs and their derived macrophages by transfecting an AAV virus containing the suicide gene Caspase9 and assisting with a chemical dimer inducer (Targeted Integration of Inducible Caspase-9 in Human iPSCs Allows Efficient in Vitro Clearance of iPSCs and iPSC-Macrophages). Small molecule treatment uses bee venom to specifically induce apoptosis and necroptosis in iPSCs via the calcium-calpain pathway, but does not affect the death of differentiated cells derived from iPSCs (Elimination of Teratogenic Human Induced Pluripotent Stem Cells by Bee Venom via Calcium-Calpain Pathway). Physical treatment methods use radiation therapy to eliminate residual undifferentiated iPSCs in iPSC-derived cardiomyocytes (Elimination of residual undifferentiated induced pluripotentstem cells (iPSCs) using irradiation for safe clinical applications of iPSC-derived cardiomyocytes).

[0004] However, all of the above methods have certain drawbacks when used to eliminate residual PSCs. Specifically, they include: Potential damage risk: Genetic modification, small molecule treatment, and physical treatment methods may potentially damage the target cells during the treatment process; Genetic treatment methods may cause potential genetic mutations in the target cells and immune responses caused by the introduction of plasmids / viruses; Complicated procedures: Genetic modification methods involve steps such as transfection plasmid design, synthesis, transfection, and positive cell screening, which consumes significant manpower and material costs during the preparation phase and increases the risk of failure due to the cumbersome procedures; Time-consuming: The cell preparation phase of genetic modification methods is time-consuming, and from plasmid design to cell line construction, it often takes more than 2 weeks. Genetic modification, small molecule treatment, and physical treatment methods often require inducing cell apoptosis or other forms of cell death during the iPSC elimination phase, which often takes hours or even days; High cost: Regardless of the above methods for eliminating residual PSCs, the PSC elimination process requires additional reagents and expenses, increasing costs. Summary of the Invention

[0005] To address the above technical issues, the present invention provides a method for removing residual pluripotent stem cells after differentiation using a balanced salt buffer solution free of calcium and magnesium ions. This method, which uses a balanced salt solution free of calcium and magnesium ions, offers a low risk of cell damage, simple steps, a short timeframe, and a low cost. This method can reduce the risk of immature teratomas and benefit patients.

[0006] The above-mentioned object of the present invention is achieved by adopting the following technical solutions:

[0007] Application of balanced salt solution without calcium and magnesium ions

[0008] First, the present invention provides the use of a balanced salt solution free of calcium ions and magnesium ions in removing residual pluripotent stem cells after differentiation of pluripotent stem cells.

[0009] Further, the balanced salt solution includes DPBS, PBS, GBSS, EBSS, HBSS, SBSS, TBSS, RBSS and / or TBS;

[0010] Optionally, the balanced salt solution is DPBS and / or PBS.

[0011] Furthermore, the pluripotent stem cells include iPSCs, ESCs and / or EPSCs;

[0012] Optionally, the pluripotent stem cells are iPSCs.

[0013] Furthermore, the balanced salt solution without calcium ions and magnesium ions is used to treat the cell system after differentiation of pluripotent stem cells at (30-40)° C. for (15-60) minutes to eliminate residual pluripotent stem cells after differentiation of pluripotent stem cells;

[0014] Optionally, the balanced salt solution without calcium ions and magnesium ions is used to treat the cell system after differentiation of pluripotent stem cells at 37° C. for 30 minutes to eliminate residual pluripotent stem cells after differentiation of pluripotent stem cells.

[0015] Furthermore, the amount of the balanced salt solution without calcium ions and magnesium ions is 0.5-3.5 mL per well of the cell culture plate;

[0016] Optionally, the amount of the balanced salt solution without calcium ions and magnesium ions is 3 mL per well of the cell culture plate.

[0017] In the present invention, the elimination of residual pluripotent stem cells after differentiation of pluripotent stem cells refers to elimination of residual pluripotent stem cells in cells obtained after differentiation of pluripotent stem cells.

[0018] In some embodiments, the cells obtained by differentiation of the pluripotent stem cells are not particularly limited, and include but are not limited to: fibrochondrocytes derived from pluripotent stem cells, osteoblasts derived from pluripotent stem cells, endothelial progenitor cells derived from pluripotent stem cells, mesenchymal stem cells derived from pluripotent stem cells, neural cells derived from pluripotent stem cells, cardiomyocytes derived from pluripotent stem cells, endothelial cells derived from pluripotent stem cells, retinal pigment epithelial cells derived from pluripotent stem cells, corneal cells derived from pluripotent stem cells, egg precursor cells derived from pluripotent stem cells, sperm precursor cells derived from pluripotent stem cells, skeletal precursor cells derived from pluripotent stem cells, blood cells derived from pluripotent stem cells, pancreatic islet cells derived from pluripotent stem cells and / or liver cells derived from pluripotent stem cells, etc.

[0019] Currently, the risk of tumorigenicity from pluripotent stem cell products primarily stems from tumorigenicity caused by differentiated and / or immature cells. Specifically, the most serious problem in pluripotent stem cell therapy is undoubtedly the formation of teratomas. Pluripotent stem cell products may form tumors or ectopic tissues in vivo. This risk is particularly acute when residual pluripotent stem cells or highly proliferative progenitor cells remain in the final product transplanted into the patient, leading to teratomas or other tumors. Therefore, researchers must prevent the development of teratomas and other tumors. This necessitates the development of a cell-safe, highly efficient, and effective PSC elimination protocol.

[0020] In the present invention, the amount of the balanced salt solution free of calcium ions and magnesium ions is not particularly limited. Any amount of the balanced salt solution free of calcium ions and magnesium ions that can eliminate a small portion, a large portion, most or all of the residual pluripotent stem cells in the cell system after differentiation of pluripotent stem cells is within the scope of protection of the present invention. Those skilled in the art can adjust the amount of the balanced salt solution free of calcium ions and magnesium ions according to actual conditions to achieve the purpose of effectively eliminating the residual pluripotent stem cells in the cell system after differentiation of pluripotent stem cells.

[0021] In the present invention, the balanced salt solution (BSS) refers to a salt solution with electrolyte content similar to that in plasma. A balanced salt solution, primarily composed of inorganic salts and glucose, is consistent with the pH and osmotic pressure of the environment in which cells grow. It maintains osmotic pressure, controls acid-base balance, and supplies the energy and inorganic salt components necessary for cell survival and metabolism. It also plays an important role in maintaining osmotic pressure, providing buffering, and adjusting the pH of the solution.

[0022] In some embodiments, the balanced salt solution free of calcium ions and magnesium ions includes, but is not limited to, Dulbecco's phosphate buffered saline (DPBS), phosphate buffered saline (PBS), Gey's balanced salt solution (GBSS), Earle's balanced salt solution (EBSS), Hanks' balanced salt solution (HBSS), Simm's balanced salt solution (SBSS), Tyrode's balanced salt solution (TBSS), Ringer's balanced salt solution (RBSS), TRIS buffer (TBS), Alsever's solution, Puck's balanced salt solution, physiological saline and / or Ringer's solution. Any balanced salt solution free of calcium ions and magnesium ions known in the art is within the scope of protection of the present invention.

[0023] In the present invention, pluripotent stem cells (PSCs) are a type of multipotent cell with the ability to self-renew and self-replicate. Under certain conditions, they can differentiate into a variety of APSCs. Pluripotent stem cells have the potential to differentiate into a variety of cell tissues, but they lose the ability to develop into a complete individual, and their developmental potential is limited.

[0024] In some embodiments, the pluripotent stem cells include but are not limited to induced pluripotent stem cells (iPSC), embryonic stem cells (ESC), expanded pluripotent stem cells (EPSC), mesenchymal stem cells (MSC) and / or parthenogenetic stem cells (PSC). Any pluripotent stem cells known in the art are within the scope of protection of the present invention.

[0025] In a specific embodiment of the present invention, a balanced salt solution (DPBS (Ca-Mg-), PBS (Ca-Mg-)) free of calcium and magnesium ions is used for the first time to remove residual pluripotent stem cells after differentiation of pluripotent stem cells, and the step of removing residual pluripotent stem cells after differentiation of pluripotent stem cells is optimized. Efficient removal of PSCs residues can be achieved only through physical changes, which is significantly superior to the PSCs residue removal method disclosed in the prior art. The present invention has developed a cell-safe and highly cost-effective iPSCs residue removal solution, which has broad application prospects in the field of pluripotent stem cell differentiation.

[0026] In a specific embodiment of the present invention, the present invention experimentally confirmed for the first time that residual pluripotent stem cells in a cell system after differentiation of pluripotent stem cells can be efficiently and successfully removed by treating the cell system after differentiation of pluripotent stem cells with a balanced salt solution (DPBS (Ca-Mg-), PBS (Ca-Mg-)) that does not contain calcium ions and magnesium ions at 37°C for 30 minutes, discarding the supernatant, and retaining the adherent cells.

[0027] A method for eliminating residual pluripotent stem cells after pluripotent stem cell differentiation

[0028] Secondly, the present invention provides a method for removing residual pluripotent stem cells after differentiation of pluripotent stem cells using a balanced salt buffer solution free of calcium ions and magnesium ions.

[0029] Furthermore, the method comprises the following step: treating a cell system after differentiation of pluripotent stem cells containing residual pluripotent stem cells in need with a balanced salt solution free of calcium ions and magnesium ions.

[0030] Further, the balanced salt solution includes DPBS, PBS, GBSS, EBSS, HBSS, SBSS, TBSS, RBSS and / or TBS;

[0031] Optionally, the balanced salt solution is DPBS and / or PBS;

[0032] Optionally, the pluripotent stem cells include iPSCs, ESCs and / or EPSCs;

[0033] Optionally, the pluripotent stem cells are iPSCs.

[0034] Furthermore, the cell system after differentiation of the pluripotent stem cells also includes cells derived from the pluripotent stem cells;

[0035] Optionally, the pluripotent stem cell-derived cells include pluripotent stem cell-derived fibrochondrocytes, pluripotent stem cell-derived osteoblasts, pluripotent stem cell-derived endothelial progenitor cells and / or pluripotent stem cell-derived mesenchymal stem cells.

[0036] In some embodiments, the pluripotent stem cell-derived cells include, but are not limited to, pluripotent stem cell-derived fibrochondrocytes, pluripotent stem cell-derived osteoblasts, pluripotent stem cell-derived endothelial progenitor cells, and / or pluripotent stem cell-derived mesenchymal stem cells. The present invention has no particular limitation on the type of pluripotent stem cell-derived cells; any cell derived from pluripotent stem cells by induction and differentiation is within the scope of the present invention.

[0037] Furthermore, the method comprises the following steps:

[0038] (1) treating a cell system after differentiation of pluripotent stem cells containing residual pluripotent stem cells in need with a balanced salt solution free of calcium ions and magnesium ions;

[0039] (2) Discard the supernatant to remove the residual pluripotent stem cells after pluripotent stem cell differentiation.

[0040] Furthermore, the treatment conditions are to treat the differentiated pluripotent stem cell system at (30-40)°C for (15-60) min;

[0041] Optionally, the treatment condition is to treat the differentiated pluripotent stem cell system at 37° C. for 30 minutes.

[0042] Furthermore, the amount of the balanced salt solution without calcium ions and magnesium ions is 0.5-3.5 mL per well of the cell culture plate;

[0043] Optionally, the amount of the balanced salt solution without calcium ions and magnesium ions is 3 mL per well of the cell culture plate.

[0044] Furthermore, the supernatant obtained after treatment with a balanced salt solution free of calcium ions and magnesium ions in step (2) contains residual pluripotent stem cells.

[0045] In some embodiments, the differentiated cell systems of residual pluripotent stem cells obtained by induced differentiation of various types of pluripotent stem cells as described above can be treated with the balanced salt solution free of calcium ions and magnesium ions as described in the present invention to effectively eliminate the residual pluripotent stem cells after differentiation of pluripotent stem cells.

[0046] In the present invention, the cell system differentiated from pluripotent stem cells comprising residual pluripotent stem cells can be cultured in a manner selected according to the actual experimental needs of those skilled in the art. For example, a cell culture plate, a cell culture dish or a cell culture flask can be selected to culture the cell system differentiated from pluripotent stem cells comprising residual pluripotent stem cells. The present invention has no particular limitation on the cell culture vessel, and any culture vessel that can be used to culture the cell system is within the scope of protection of the present invention.

[0047] In some embodiments, cell culture vessels can be categorized by material as glass or plastic. Glass has the advantage of being easy to grow cells, clean, and reuse. Plastic has the advantage of being inexpensive, disposable, and reducing contamination risks. Currently, polystyrene (PS) is the most commonly used plastic in laboratories. Its low cost and inert chemical properties make it an excellent choice for disposable culture surfaces.

[0048] In some embodiments, there are three common types of cell culture vessels: culture flasks, culture dishes, and multi-well culture plates.

[0049] Petri dishes are shallow and easy to handle. They are commonly used for cell growth and propagation. They are available in a variety of sizes (single-well or multi-well), named according to the diameter of their base. Common sizes include 6 cm and 10 cm. Compared to culture flasks, selecting individual cell colonies from a Petri dish is easier and more convenient. However, due to the larger opening, the risk of contamination is also higher.

[0050] Culture flasks are named by their surface area, such as T25, T75, and T175. The numbers represent the bottom area of ​​the flask, and T25 is 25 cm 2 The bottle body design can be divided into straight neck or curved neck. Curved neck culture bottles are easier to operate, and the bottom surface can be touched with a pipette or long gun tip. It also prevents the bottle cap from contacting the culture medium, reducing liquid splashing and reducing the risk of contamination. Depending on the needs of different cells, the bottle cap design can be divided into two types: filter membrane and seal. For cells that require CO2 ventilation under culture conditions, it is recommended to use a breathable filter membrane model.

[0051] Multi-well culture plates are also commonly made of plastic and are used in various detection experiments, such as cell cloning, scratch, and cytotoxicity experiments. Commonly used culture plates are divided into 6-well, 12-well, 24-well, and 96-well types according to the number of wells.

[0052] Application of balanced salt solution without calcium ions and magnesium ions in the preparation of cell products

[0053] In addition, the present invention also provides the use of a balanced salt solution free of calcium ions and magnesium ions in the preparation of a cell product derived from pluripotent stem cells.

[0054] Further, the balanced salt solution includes DPBS, PBS, GBSS, EBSS, HBSS, SBSS, TBSS, RBSS and / or TBS;

[0055] Optionally, the balanced salt solution is DPBS and / or PBS.

[0056] Furthermore, the pluripotent stem cells include iPSCs, ESCs and / or EPSCs;

[0057] Optionally, the pluripotent stem cells are iPSCs.

[0058] Furthermore, the balanced salt solution without calcium ions and magnesium ions is used to treat the cell system after differentiation of pluripotent stem cells at (30-40)° C. for (15-60) minutes to eliminate residual pluripotent stem cells after differentiation of pluripotent stem cells;

[0059] Optionally, the balanced salt solution without calcium ions and magnesium ions is used to treat the cell system after differentiation of pluripotent stem cells at 37° C. for 30 minutes to eliminate residual pluripotent stem cells after differentiation of pluripotent stem cells.

[0060] Furthermore, the amount of the balanced salt solution without calcium ions and magnesium ions is 0.5-3.5 mL per well of the cell culture plate;

[0061] Optionally, the amount of the balanced salt solution without calcium ions and magnesium ions is 3 mL per well of the cell culture plate.

[0062] In some embodiments, the cells derived from pluripotent stem cells are not particularly limited, and include but are not limited to: fibrochondrocytes derived from pluripotent stem cells, osteoblasts derived from pluripotent stem cells, endothelial progenitor cells derived from pluripotent stem cells, mesenchymal stem cells derived from pluripotent stem cells, neural cells derived from pluripotent stem cells, cardiomyocytes derived from pluripotent stem cells, endothelial cells derived from pluripotent stem cells, retinal pigment epithelial cells derived from pluripotent stem cells, corneal cells derived from pluripotent stem cells, egg precursor cells derived from pluripotent stem cells, sperm precursor cells derived from pluripotent stem cells, skeletal precursor cells derived from pluripotent stem cells, blood cells derived from pluripotent stem cells, pancreatic islet cells derived from pluripotent stem cells and / or liver cells derived from pluripotent stem cells, etc.

[0063] Compared with the prior art, the present invention has the following beneficial effects:

[0064] The present invention is the first to apply balanced salt solutions (DPBS (Ca-Mg-), PBS (Ca-Mg-)) that are free of calcium and magnesium ions to remove residual pluripotent stem cells after differentiation. This solution optimizes the PSC removal process and, after differentiation is complete, uses only the balanced salt solution commonly used in cell culture, which is less damaging to cells, to minimize the risk of potential cell damage. This solution achieves efficient removal of residual PSCs through physical changes alone, and its efficiency is significantly superior to conventional PSC removal methods disclosed in the prior art. Specifically, the following are the steps to remove residual PSCs:

[0065] (1) Compared with the methods disclosed in the prior art, the method provided by the present invention for removing residual pluripotent stem cells after differentiation of pluripotent stem cells is simple to operate and only requires soaking in the balanced salt solution;

[0066] (2) Compared with the methods disclosed in the prior art, the method provided by the present invention for removing residual pluripotent stem cells after differentiation of pluripotent stem cells is time-consuming, and the entire removal process does not exceed 1 hour;

[0067] (3) Compared with the methods disclosed in the prior art, the method provided by the present invention for removing residual pluripotent stem cells after differentiation of pluripotent stem cells is low-cost and only requires a low-cost balanced salt solution (for example, only 3 mL of balanced salt solution is required to remove PSCs from each well of a 6-well plate, and the calculated price of 500 mL of balanced salt solution is approximately RMB 100, so only RMB 0.6 is required to remove PSCs from each well of a 6-well plate). BRIEF DESCRIPTION OF THE DRAWINGS

[0068] Figure 1 Figure 2 shows the response of PSCs and MSCs to DPBS (Ca-Mg-) at room temperature, where: Panel A: iPSCs-001-5, Panel B: iPSCs-006-1, Panel C: EPSCs-006-1, Panel D: EPSCs-001-5, Panel E: iMSCs.

[0069] Figure 2 The results of the responses of PSCs and MSCs to DPBS (Ca-Mg-) at 37°C are shown, among which, Panel A: iPSCs-001-5, Panel B: iPSCs-006-1, Panel C: EPSCs-006-1, Panel D: EPSCs-001-5, Panel E: iMSCs;

[0070] Figure 3 The cell morphology of iMSCs treated with DPBS (Ca-Mg-) at room temperature and 37°C and then rewarmed at 37°C. The left picture is at room temperature, and the right picture is at 37°C.

[0071] Figure 4Figures 2A and 2B show the cytoskeleton morphology of PSCs and iMSCs after treatment with DPBS (Ca-Mg-) at room temperature and 37°C. Figure A shows the cytoskeleton morphology of PSCs and iMSCs after treatment with DPBS (Ca-Mg-) at room temperature. Figure B shows the cytoskeleton morphology of PSCs and iMSCs after treatment with DPBS (Ca-Mg-) at 37°C.

[0072] Figure 5 Statistical graphs of cell death of PSCs and iMSCs after treatment with DPBS (Ca-Mg-) at room temperature and 37°C, wherein, Panel A: Cell death of PSCs and iMSCs after treatment with DPBS (Ca-Mg-) at room temperature, Panel B: Cell death of PSCs and iMSCs after treatment with DPBS (Ca-Mg-) at 37°C;

[0073] Figure 6 Figures 1 and 2 are the crystal violet staining results of PSCs and iMSCs after treatment with DPBS (Ca-Mg-), wherein: Panel A: Crystal violet staining results of iPSCs-001-5 and iMSCs after treatment with DPBS (Ca-Mg-) at room temperature; Panel B: Crystal violet staining results of iPSCs-001-5 and iMSCs after treatment with DPBS (Ca-Mg-) at 37°C; Panel C: Crystal violet staining results of iPSCs-006-1 after treatment with DPBS (Ca-Mg-) at room temperature and 37°C; Panel D: Crystal violet staining results of EPSCs-006-1 after treatment with DPBS (Ca-Mg-) at room temperature and 37°C; Panel E: Crystal violet staining results of EPSCs-001-5 after treatment with DPBS (Ca-Mg-) at room temperature and 37°C; Figure 7 Figures show the establishment of a co-culture system between PSCs and differentiated cells and the results of light microscopy observation after DPBS (Ca-Mg-). Figure A shows the establishment of a co-culture system between iPSCs and iMSCs and the results of light microscopy observation after DPBS (Ca-Mg-). Figure B shows the establishment of a co-culture system between iPSCs and iOBs, iFCs, and iEPCs and the results of light microscopy observation after DPBS (Ca-Mg-).

[0074] Figure 8 The expression of EGFP (green fluorescence) in each group before and after DPBS (Ca-Mg-) treatment, wherein, Figure A: the detection results of the expression of EGFP (green fluorescence) in each group before and after DPBS (Ca-Mg-) treatment by laser confocal microscopy, Figure B: the detection results of the expression of EGFP (green fluorescence) in each group before and after DPBS (Ca-Mg-) treatment by flow cytometry;

[0075] Figure 9Figure 1 shows the cell cycle and qPCR results after iPSCs were cleared with DPBS (Ca-Mg-) in the co-culture system of iPSCs and iMSCs. Figure 1 shows the cell cycle and qPCR results after iPSCs were cleared with DPBS (Ca-Mg-) in the co-culture system of iPSCs and iMSCs. Figure 1 shows the cell cycle and qPCR results after iPSCs were cleared with DPBS (Ca-Mg-) in the co-culture system of iPSCs and iMSCs. DETAILED DESCRIPTION

[0076] The present invention will be further described below with reference to specific embodiments. The specific embodiments are intended only to explain the present invention and are not to be construed as limiting the present invention. Those skilled in the art will appreciate that various changes, modifications, substitutions, and variations may be made to these embodiments without departing from the principles and intent of the present invention. The scope of the present invention is defined by the claims and their equivalents.

[0077] The reagents and raw materials used in the present invention are readily available to those skilled in the art and, unless otherwise specified, can be obtained from commercial sources. Experimental methods not specifying specific conditions in the present invention are generally carried out under conventional conditions or conditions recommended by the manufacturer. In particular, the following examples are only used to illustrate the present invention and should not be construed to limit the scope of the present invention in any way.

[0078] Example 1: Process of differentiation from PSCs to mesenchymal stem cells (MSCs), osteoblasts, fibrochondrocytes, and endothelial progenitor cells

[0079] 1.1 MSCs: PSCs were induced to differentiate into MSCs using the method described in Chinese patent publication CN115094031A.

[0080] 1.2 Osteoblasts: PSCs were induced to differentiate into osteoblasts using the method described in Chinese Patent Publication CN117535234A.

[0081] 1.3 Fibrochondrocytes: PSCs were induced to differentiate into fibrochondrocytes using the method described in Chinese Patent Publication CN117126801A.

[0082] 1.4 Endothelial progenitor cells: PSCs were induced to differentiate into endothelial progenitor cells using the method described in Chinese Patent Publication CN108384746A.

[0083] Example 2 Exploration of iPSCs Clearing Conditions

[0084] The present invention first uses iPSCs and iMSCs to explore the clearance conditions.

[0085] 2.1 Exploration of room temperature conditions

[0086] 2.1.1 Cell morphology recording

[0087] PSCs (if no special records are provided, the PSCs used in Section 2.1 are the four cell lines iPSCs-001-5, iPSCs-006-1, EPSCs-001-5, and EPSCs-006-1, which were prepared by our company. iPSCs were prepared using the method described in Chinese Patent Publication CN109913494A, and EPSCs were prepared by culturing EPSCs in EPSC medium for 10 generations) and iMSCs (prepared by our company, prepared according to the method described in Chinese Patent Publication CN115094031A) were plated in 6-well plates coated with Matrigel. The experiment was performed after the growth confluence reached 70%, the supernatant was aspirated, and 1 mL of E8 medium (for iPSCs, TeSR TM -E8 TM , Catalog # 05990, Stemcell, USA) / EPSCs medium (for EPSCs, provided by Chengnuo Medical) / MSCs medium (for iMSCs, αMEM (11900073, ThermoFisher Scientific, USA) + 5% Ultra GROTM-Advanced Cell Nutrition Supplement (hPL, HPCFDCRL50, Helios, USA)), DPBS (Ca-Mg-), DPBS (Ca+Mg+), PBS (Ca-Mg-) were added to each well to wash the cells once. Then, 2 mL of E8 medium (for iPSCs) / EPSCs medium (for EPSCs) / MSCs medium (for iMSCs), 2 mL of DPBS (Ca-Mg-), 2 mL of DPBS (Ca+Mg+), and 2 mL of PBS (Ca-Mg-) were added to each well and incubated at room temperature for 15 minutes, 30 minutes, 60 minutes, and 120 minutes. The cell morphology at each time point was recorded under a microscope. Figure 1 iMSCs-MSCs were treated with MSCs culture medium and DPBS (Ca-Mg-) at room temperature for 120 minutes and then incubated at 37°C for 60 minutes. Cell morphology was recorded under a microscope. Figure 3 ).

[0088] 2.1.2 Phalloidin staining

[0089] iPSCs-001-5 and iMSCs were plated in Matrigel-coated 12-well plates. Experiments were performed after growth reached 70% confluence. The supernatant was aspirated, and the cells were washed once per well with 1 mL of E8 medium (for iPSCs) / MSCs medium (for iMSCs), DPBS (Ca-Mg-), DPBS (Ca+Mg+), and PBS (Ca-Mg-). Then, 2 mL of E8 medium (for iPSCs) / MSCs medium (for iMSCs), 2 mL of DPBS (Ca-Mg-), 2 mL of DPBS (Ca+Mg+), and 2 mL of PBS (Ca-Mg-) were added to each well and incubated at room temperature for 30 minutes. The supernatant was aspirated, and the cells were fixed with 4% paraformaldehyde solution at room temperature for 20 minutes and washed three times with PBS (Ca-Mg-). The prepared phalloidin working solution was added and incubated at room temperature for 60 minutes, followed by washing three times with PBS (Ca-Mg-). The cells were then stained with DAPI solution for 5 minutes at room temperature and washed three times with PBS (Ca-Mg-). Observe, photograph and record under laser confocal microscope ( Figure 4 ).

[0090] 2.1.3 Trypan blue staining and crystal violet staining

[0091] PSCs and iMSCs were plated in Matrigel-coated 6-well plates. Experiments were performed after growth reached 70% confluence. The supernatant was aspirated, and the cells were washed once per well with 1 mL of E8 medium (for iPSCs), EPSCs medium (for EPSCs), MSCs medium (for iMSCs), DPBS (Ca-Mg-), DPBS (Ca+Mg+), and PBS (Ca-Mg-). Then, 2 mL of E8 medium (for iPSCs), EPSCs medium (for EPSCs), MSCs medium (for iMSCs), DPBS (Ca-Mg-), DPBS (Ca+Mg+), and PBS (Ca-Mg-) were added to each well and incubated at room temperature for 60 minutes. The cells were washed three times with E8 medium (for iPSCs), MSCs medium (for iMSCs), 2 mL of DPBS (Ca-Mg-), 2 mL of DPBS (Ca+Mg+), and 2 mL of PBS (Ca-Mg-). 4% paraformaldehyde was added to each well and incubated at room temperature for 30 minutes. Figure 6 At the same time, the cell washes from the DPSC (Ca-Mg-) group and the PSC (Ca-Mg-) group in iPSCs-001-5 were collected and centrifuged at 1000r for 5 minutes. The supernatant was discarded and incubated with 0.4% trypan blue solution at room temperature for 5 minutes. The cells were counted under a microscope and statistically analyzed ( Figure 5 ).

[0092] 2.1.4 Experimental results at room temperature

[0093] The present invention first used iPSCs-001-5 and iMSCs cell lines to explore room temperature conditions. After treating iPSCs-001-5 with E8 medium and DPBS (Ca+Mg+) at room temperature for 15 minutes, 30 minutes, 60 minutes, and 120 minutes, the cell morphology did not change significantly. After treating with DPBS (Ca-Mg-) and PBS (Ca-Mg-) at room temperature for 30 minutes, iPSCs-001-5 cells began to shrink and the cell edges became rounded, and this phenomenon gradually worsened over time ( Figure 1 After being treated with DPBS (Ca-Mg-) at room temperature for 120 minutes, iPSCs-001-5 cells were observed to be detached from the extracellular matrix (ECM) in sheets ( Figure 1 Repeated experiments with three other PSCs cell lines also reproduced this result ( Figure 1 After iMSCs were treated with MSCs culture medium, DPBS (Ca-Mg-), DPBS (Ca+Mg+), and PBS (Ca-Mg-) at room temperature for 120 minutes, the cells in each group shrank slightly, but the cell morphology was basically the same as before treatment.

[0094] The phalloidin staining experiment found that after iPSCs-001-5 was treated with DPBS (Ca-Mg-) and PBS (Ca-Mg-) at room temperature for 30 minutes, the cytoskeleton of iPSCs-001-5 was significantly shrunk ( Figure 4 However, the cytoskeleton of iPSCs-001-5 treated with E8 medium and DPBS (Ca+Mg+) and iMSCs treated with MSCs medium, DPBS (Ca-Mg-), DPBS (Ca+Mg+), and PBS (Ca-Mg-) did not change significantly ( Figure 4 ).

[0095] The results of trypan blue staining showed that the mortality rate of iPSCs-001-5 was about 30% in the cell wash solution collected after iPSCs-001-5 was treated with DPBS (Ca-Mg-) and PBS (Ca-Mg-) at room temperature for 60 minutes ( Figure 5 ).

[0096] The crystal violet staining results showed that iPSCs-001-5 treated with DPBS (Ca-Mg-) and PBS (Ca-Mg-) were completely cleared and crystal violet was not stained. Except for these two wells, the cells in the remaining wells were stained normally, indicating that the cells in other groups were not affected ( Figure 6 ).

[0097] The above results suggest that treatment of PSCs with DPBS (Ca-Mg-) and PBS (Ca-Mg-) for 60 min at room temperature can effectively eliminate PSCs without affecting the adhesion of iMSCs.

[0098] 2.2 Exploration of 37°C conditions

[0099] 2.2.1 Cell morphology recording

[0100] PSCs (unless otherwise noted, the PSCs used in Section 2.2 were the four cell lines iPSCs-001-5, iPSCs-006-1, EPSCs-001-5, and EPSCs-006-1, the sources of which were described above) and iMSCs (the sources of which were described above) were plated in 6-well plates coated with Matrigel. Experiments were performed after growth confluence reached 70%, the supernatant was aspirated, and the cells were washed once per well with 1 mL of E8 medium (for iPSCs) / EPSCs medium (for EPSCs) / MSCs medium (for iMSCs), 2 mL of DPBS (Ca-Mg-), 2 mL of DPBS (Ca+Mg+), and 2 mL of PBS (Ca-Mg-). Then, 2 mL of E8 medium (for iPSCs) / EPSCs medium (for EPSCs) / MSCs medium (for iMSCs), 2 mL of DPBS (Ca-Mg-), 2 mL of DPBS (Ca+Mg+), and 2 mL of PBS (Ca-Mg-) were added to each well and incubated at room temperature for 15 minutes, 30 minutes, 60 minutes, and 120 minutes. Cell morphology at each time point was recorded under a microscope. Figure 2 iMSCs-MSCs were treated with MSCs culture medium and DPBS (Ca-Mg-) at room temperature for 120 minutes and then incubated at 37°C for 60 minutes. Cell morphology was recorded under a microscope. Figure 3 ).

[0101] 2.2.2 Phalloidin staining

[0102] iPSCs-001-5 and iMSCs were plated in a 12-well plate coated with Matrigel. The experiment was carried out after the growth confluence reached 70%, the supernatant was aspirated, and the cells were washed once with 1 mL of E8 medium (for iPSCs) / MSCs medium (for iMSCs) and 2 mL of DPBS (Ca-Mg-) per well. Then 2 mL of E8 medium (for iPSCs) / MSCs medium (for iMSCs) and 2 mL of DPBS (Ca-Mg-) were added to each well and incubated at room temperature for 15 minutes. The supernatant was aspirated, fixed with 4% paraformaldehyde solution at room temperature for 20 minutes, and washed three times with PBS (Ca-Mg-). The prepared phalloidin working solution was added and incubated at room temperature for 60 minutes, and washed three times with PBS (Ca-Mg-). The prepared DAPI staining solution was used to incubate at room temperature for 5 minutes, and washed three times with PBS (Ca-Mg-). Observe, photograph, and record under laser confocal microscopy ( Figure 4 ).

[0103] 2.2.3 Trypan blue staining and crystal violet staining

[0104] PSCs and iMSCs were plated in Matrigel-coated 6-well plates. Experiments were performed after growth reached 70% confluence. The supernatant was aspirated, and the cells were washed once per well with 1 mL of E8 medium (for iPSCs), EPSCs medium (for EPSCs), or MSCs medium (for iMSCs), 2 mL of DPBS (Ca-Mg-), 2 mL of DPBS (Ca+Mg+), and 2 mL of PBS (Ca-Mg-). Then, 2 mL of E8 medium (for iPSCs), EPSCs medium (for EPSCs), or MSCs medium (for iMSCs), 2 mL of DPBS (Ca-Mg-), 2 mL of DPBS (Ca+Mg+), and 2 mL of PBS (Ca-Mg-) were added to each well and incubated at room temperature for 30 minutes. The cells were washed three times with E8 medium (for iPSCs), MSCs medium (for iMSCs), DPBS (Ca-Mg-), DPBS (Ca+Mg+), and PBS (Ca-Mg-). 4% paraformaldehyde was added to each well and incubated at room temperature for 30 minutes. Figure 3 At the same time, the cell washes from the DPSC (Ca-Mg-) group and the PSC (Ca-Mg-) group in iPSCs-001-5 were collected and centrifuged at 1000r for 5 minutes. The supernatant was discarded and incubated with 0.4% trypan blue solution at room temperature for 5 minutes. The cells were counted under a microscope and statistically analyzed ( Figure 5 ).

[0105] 2.2.4 Experimental results at 37°C

[0106] To further clarify the optimal experimental and treatment conditions, the present invention further used iPSCs-001-5 and iMSCs cell lines to explore 37°C conditions. After treating iPSCs-001-5 with E8 medium and DPBS (Ca+Mg+) at 37°C for 15 minutes, 30 minutes, 60 minutes, and 120 minutes, the cell morphology did not change significantly. After being treated with DPBS (Ca-Mg-) and PBS (Ca-Mg-) at 37°C for 15 minutes, iPSCs-001-5 cells began to shrink and the cell edges became rounded, and this phenomenon gradually worsened over time ( Figure 2 After being treated with DPBS (Ca-Mg-) at room temperature for 60 minutes, iPSCs-001-5 cells were observed to be detached from the extracellular matrix (ECM) in sheets ( Figure 2 Repeated experiments with three other PSCs cell lines also reproduced this result ( Figure 2 After iMSCs were treated with MSCs culture medium, DPBS (Ca-Mg-), DPBS (Ca+Mg+), and PBS (Ca-Mg-) at 37°C for 120 minutes, the cell morphology of each group was basically the same as before treatment.

[0107] The phalloidin staining experiment found that after iPSCs-001-5 was treated with DPBS (Ca-Mg-) at 37℃ for 15 minutes, the cytoskeleton of iPSCs-001-5 was significantly shrunk ( Figure 4 However, the cytoskeleton of iPSCs-001-5 treated with E8 medium and iMSCs treated with MSCs medium and DPBS (Ca-Mg-) did not change significantly ( Figure 4 ).

[0108] The results of trypan blue staining showed that the mortality rate of iPSCs-001-5 was about 25% in the cell wash solution collected after iPSCs-001-5 was treated with DPBS (Ca-Mg-) and PBS (Ca-Mg-) at room temperature for 30 minutes ( Figure 5 ).

[0109] The crystal violet staining results showed that iPSCs-001-5 treated with DPBS (Ca-Mg-) and PBS (Ca-Mg-) were completely cleared and crystal violet was not stained. Except for these two wells, the cells in the remaining wells were stained normally, indicating that the cells in other groups were not affected ( Figure 6 ).

[0110] These results suggest that treating PSCs with DPBS (Ca-Mg-) and PBS (Ca-Mg-) for 30 minutes at 37°C effectively eliminates PSCs without affecting the attachment of iMSCs. Combined with the experimental results in Sections 2.1 and 2.2, the present invention adopted DPBS (Ca-Mg-) treatment for 30 minutes at 37°C as the final experimental condition for subsequent experiments.

[0111] 2.3 Validation of the co-culture system of PSCs and PSC-differentiated cells

[0112] 2.3.1 Construction of iPSCs-001-5-EGFP cell line

[0113] A lentiviral vector carrying the EGFP gene and Puro resistance was designed and constructed, and lentiviral particles were produced using packaging cells. Next, the lentiviral particles were infected with iPSCs-001-5 cells using Polybrane. Under appropriate culture conditions, Puro was added to screen for cell lines stably expressing EGFP. Finally, the expression efficiency and stability of EGFP were tested and verified using fluorescence microscopy and flow cytometry.

[0114] 2.3.2 Co-culture of iPSCs-001-5-EGFP and PSC-derived Differentiated Cells

[0115] To further clarify whether the above experiments can be implemented in a co-culture system (actual application system), the present invention further used the iPSCs-001-5-EGFP cell line to co-culture with iMSCs, iPSCs-derived fibrochondrocytes (iFCs), osteoblasts (iOBs), and endothelial progenitor cells (iEPCs) to verify the clearance effect.

[0116] First, PSCs were plated at 500,000 cells / well and 1.5 million cells / well in a 6-well plate coated with Matrigel. After 24 hours, iMSCs, iFCs, iOBs, and iEPCs were added at 1.5 million cells / well and 500,000 cells / well, respectively. Incubation was continued at room temperature for 6 hours. Once most cells had attached to the wall, the co-culture system was successfully established. 2.3.3 Clearance Verification of the Co-culture System of iPSCs-001-5-EGFP and PSC-derived Differentiated Cells

[0117] Wash cells once with 1 mL of DPBS (Ca-Mg-) per well (6-well plate). Add 2 mL of DPBS (Ca-Mg-) per well and treat the cells according to the previous conditions (incubation at 37°C for 30 min). After treatment, wash each well three times with DPBS (Ca-Mg-). Observe the proportion of positive cells using confocal microscopy and photograph the remaining cells.

[0118] Afterwards, the clearing effect of DPBS (Ca-Mg-) was studied by flow cytometry, qPCR and cell cycle detection on cleared and non-cleared cells.

[0119] 2.3.4 Validation results of the co-culture system of PSCs and PSC-differentiated cells

[0120] The present invention successfully achieved the establishment of a co-culture system of iPSCs, iMSCs, iFCs, iOBs, and iEPCs ( Figure 4 B, Figure 5 After incubation in DPBS (Ca-Mg-) at 37°C for 30 min (the amount of DPBS (Ca-Mg-) was 2 mL per well of the culture plate), it was observed that some cells in each group had shiny edges and shrunk into a round shape, while some cells still adhered to the bottom of the dish ( Figure 7 The morphology of adherent cells is different from the conventional morphology of iPSCs and is similar to that of differentiated cells. After washing three times with DPBS (Ca-Mg-), only cells with a morphology similar to that of differentiated cells remain at the bottom of the dish ( Figure 7 ).

[0121] Subsequently, before DPBS (Ca-Mg-) treatment, laser confocal microscopy results showed that some cells in each group still expressed EGFP (green fluorescence). After DPBS (Ca-Mg-) treatment, most of the EGFP+ cells were successfully eliminated ( Figure 8 Flow cytometry results also supported this result ( Figure 8 ).

[0122] Subsequently, qPCR results showed that iPSCs expressed higher levels of pluripotent stem cell genes (OCT4, SOX2, Nanog) compared with iMSCs ( Figure 9 The expression levels of pluripotent stem cell genes (OCT4, SOX2, Nanog) in each iPSCs and iMSCs co-culture system were between those of iPSCs and iMSCs ( Figure 9 After DPBS (Ca-Mg-) removal, there was no statistical difference in the expression of pluripotent stem cell genes (OCT4, SOX2, Nanog) between the removal group and iMSCs ( Figure 9 ). This indicates that, based on gene expression levels, cells with high expression of pluripotent stem cell genes were successfully eliminated by DPBS (Ca-Mg-).

[0123] Subsequently, the cell cycle results showed that the proportion of G0 / G1 phase cells in the iMSCs group was higher than that in the iPSCs group. The proportion of G0 / G1 phase cells in each iPSCs and iMSCs co-culture system was between the iMSCs group and the iPSCs group. After DPBS (Ca-Mg-) removal, there was no statistical difference in the proportion of G0 / G1 phase cells between the removal group and the iMSCs group ( Figure 9 ).

Claims

1. A method for removing residual pluripotent stem cells after differentiation of pluripotent stem cells using a balanced salt buffer solution free of calcium ions and magnesium ions, characterized in that: The method comprises the following steps: (1) treating a cell system after differentiation of pluripotent stem cells containing residual pluripotent stem cells in need with a balanced salt solution free of calcium ions and magnesium ions; (2) discarding the supernatant to remove residual pluripotent stem cells after pluripotent stem cell differentiation; Described balanced salt solution is DPBS and / or PBS; The pluripotent stem cells are iPSCs; The treatment conditions are to treat the differentiated cell system of pluripotent stem cells at (30-40)° C. for (15-60) minutes.

2. The method according to claim 1, characterized in that The cell system after differentiation of the pluripotent stem cells also includes cells derived from the pluripotent stem cells.

3. The method according to claim 2, characterized in that The cells derived from pluripotent stem cells include fibrochondrocytes derived from pluripotent stem cells, osteoblasts derived from pluripotent stem cells, endothelial progenitor cells derived from pluripotent stem cells and / or mesenchymal stem cells derived from pluripotent stem cells.

4. The method according to claim 1, wherein The treatment condition is to treat the cell system after differentiation of pluripotent stem cells at 37° C. for 30 minutes.

5. The method according to claim 1, wherein The amount of the balanced salt solution without calcium ions and magnesium ions is 0.5-3.5 mL per well of the cell culture plate.

6. The method according to claim 5, characterized in that The amount of the balanced salt solution without calcium ions and magnesium ions is 3 mL per well of the cell culture plate.

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