A method for preparing mesenchymal stem cells differentiated from embryonic stem cells expressing PGE-2 anchored on a membrane and application thereof

Abstract

The application discloses a preparation method of mesenchymal stem cells differentiated from embryo stem cells and anchored to membranes and expressing PGE-2 and application thereof. The preparation method comprises the following steps: (1) preparing a membrane-anchored PGE-2 overexpressing hESC cell line hESC OE‑PGE‑2 ; (2) differentiating hESC OE‑PGE‑2 cells into hESC-MSCs OE‑PGE‑2 cells. The preparation method differentiates hESC into MSCs by adding growth factors such as EGF and bFGF, and is an important source of MSC seed cells, solves the problem of the immunogenicity limitation of embryo stem cells, solves the problem of the instability of the genome of primary MSCs in terms of source, and has the advantages of non-invasive acquisition of seed cells and good editability, repeatability and stability of the seed cells. The prepared hESC-MSCs OE‑PGE‑2 do not need to be extracted invasively, can be better controlled in quality, can avoid gene mutation to a certain extent, have strong proliferation capacity, and are anchored to membranes and continuously express PGE-2 by using gene editing technology, so that the treatment effect of anti-hepatic fibrosis is greatly improved.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, specifically to a method for preparing mesenchymal stem cells derived from embryonic stem cells that express PGE-2 via membrane anchoring, and its application. Background Technology

[0002] Liver fibrosis is a serious disease characterized by permanent scarring following hepatocellular damage and accompanied by high mortality. It is typically caused by severe external trauma, autoimmune responses, viral hepatitis, and adverse drug reactions. Studies have shown that the progression of liver fibrosis can be roughly divided into three stages: In the first stage, liver injury or other harmful stimuli cause hepatic stellate cells to transition from a quiescent to an activated state, leading to fibroblast activation and excessive production of extracellular matrix (ECM). In the second stage, activated ECM undergoes remodeling of structures such as liver lobules, while T cells and intrahepatic macrophages are activated, producing various cytokines. In the third stage, the damaging factors persist, fibroblasts continue to be activated, producing even more ECM. Cytokines continuously induce tissue inflammation and collagen overexpression, leading to continuous ECM deposition and scar tissue formation. Liver fibrosis gradually develops, ultimately resulting in cirrhosis and loss of liver function.

[0003] In recent years, although advancements in technology have led to some progress in the research of drugs for treating liver fibrosis, the pathogenesis of liver fibrosis remains incompletely understood, thus effective treatment methods are still lacking. Currently, the main treatment strategies for liver fibrosis include anti-inflammatory, anti-fibrotic, and anti-oxidative approaches, with treatment methods primarily including drug therapy, surgery, and liver transplantation. However, drug therapy has significant side effects, and liver transplantation, currently the most effective treatment for liver fibrosis, is limited in its application due to drawbacks such as a lack of donated organ resources, rejection reactions, infections, complications, and high costs.

[0004] Stem cell therapy has developed rapidly in recent years, with mesenchymal stem cells (MSCs) being ideal seed cells for tissue regeneration and clinical translational therapy. MSCs are widely available, with bone marrow and adipose tissue being the most common sources. However, these currently used MSCs generally suffer from the following problems: ① Extraction requires invasive techniques, such as bone marrow aspiration and fat extraction, which is a burden on donors; ② While most are commercially available, due to differences in donors, each MSC has a different genome and genetic background, leading to heterogeneity in seed cells. For patients requiring multiple MSC transplants, the treatment effect may not meet expectations, and there is a certain risk of genetic mutations; ③ The proliferation capacity of primary MSCs is limited by the growth rate and senescence / apoptosis that occurs during passage culture, making it unable to cope with sudden shortages of seed cells; ④ Gene editing efficiency in MSCs is low. Therefore, these bottlenecks significantly limit the application of MSCs in precision medicine, prompting the search for new sources of MSC seed cells. Although human embryonic stem cells (hESCs) have unlimited self-renewal and differentiation potential, their clinical use is greatly limited due to issues such as immunogenicity, and therefore there are few reports of their use in treating liver fibrosis. Summary of the Invention

[0005] The purpose of this invention is to address the technical problems of mesenchymal stem cells (MSCs) used in current stem cell therapy for anti-fibrosis, such as the need for invasive extraction techniques, limited proliferation capacity, and impaired differentiation capacity during culture and passage, which leads to a decline in function with the age of the donor, thus limiting the therapeutic effect. The invention provides a method for preparing mesenchymal stem cells derived from embryonic stem cells that express PGE-2 on a membrane and its application.

[0006] The technical solution adopted in this invention is as follows: A method for preparing mesenchymal stem cells derived from embryonic stem cells that express PGE-2 via membrane anchoring includes the following steps: (1) Preparation of membrane-anchored PGE-2 overexpressing hESC cell line hESC OE-PGE-2 ; (2) hESC OE-PGE-2 Cell differentiation into hESC-MSCs OE-PGE-2 cell.

[0007] Furthermore, the method for preparing mesenchymal stem cells derived from membrane-anchored PGE-2-expressing embryonic stem cells described above includes the following steps in step (1): S1. A membrane-anchored PGE-2 cloning vector was constructed using cloning technology and packaged into a lentivirus to obtain a membrane-anchored lentivirus overexpressing PGE-2; S2. Infection of embryonic stem cells with lentiviruses that overexpress PGE-2 via membrane anchoring; S3. Stable membrane-anchored hESCs were obtained by flow cytometry sorting. OE-PGE-2 Cell lines.

[0008] Furthermore, the method for preparing mesenchymal stem cells derived from membrane-anchored PGE-2-expressing embryonic stem cells described above includes the following three stages in step (2): In the first stage, the feeder cells were removed and hESC was performed in Matri-Gel-coated cell culture dishes. OE-PGE-2 Cell culture, removing the Matri-Gel coating condition in the culture system, and inducing hESCs OE-PGE-2 Cellular self-differentiation; In the second stage, trypsin was used to differentiate the hESCs. OE-PGE-2 Cells are digested into single cells and cultured, which promotes the proliferation of mesenchymal stem cell-like cells; In the third stage, cells were passaged every other day to enrich mesenchymal stem cell-like cells. The highly enriched mesenchymal stem cell-like cells were then sorted by flow cytometry and labeled with CD24. - / CD105 + MSCs were sorted and purified, and then cultured as single clones in 96-well plates to expand MSCs and establish hESC-MSCs. OE-PGE-2 Tie.

[0009] Furthermore, in the method for preparing mesenchymal stem cells derived from membrane-anchored PGE-2-expressing embryonic stem cells described above, the induction time in the first stage is 3 days.

[0010] Furthermore, in the method for preparing mesenchymal stem cells derived from membrane-anchored PGE-2-expressing embryonic stem cells described above, the culture and proliferation time in the second stage is 4 days.

[0011] Furthermore, in the above-described method for preparing mesenchymal stem cells derived from embryonic stem cells that express PGE-2 on a membrane, the components of the culture medium used in the second stage of culture include: RPMI-1640 (basal medium), 20% KOSR (gene knockout serum replacement medium), 10 ng / mL bFGF (basal fibroblast growth factor), 20 ng / mL PDGFAB (platelet-derived growth factor AB), and 10 ng / mL EGF (epidermal growth factor).

[0012] Furthermore, in the method for preparing mesenchymal stem cells derived from membrane-anchored PGE-2-expressing embryonic stem cells described above, the third stage of culture time is 7-14 days.

[0013] The application of mesenchymal stem cells derived from embryonic stem cells that express PGE-2 on a membrane, as described above, is for use in the preparation of drugs for treating liver fibrosis.

[0014] The beneficial effects of this invention are as follows: 1. hESC-MSCs in this invention OE-PGE-2 It eliminates the need for invasive extraction, overcoming the problem that current MSC extraction methods require invasive techniques and greatly reducing the suffering of donors.

[0015] 2. hESC-MSCs in this invention OE-PGE-2 Differentiation originates from the same hESC with a uniform and stable genome, which allows for better quality control of seed cells and avoids gene mutations to a certain extent. This overcomes the problems of heterogeneity of seed cells, unsatisfactory therapeutic effects, and certain risks of genetic mutations associated with currently used MSCs, thus improving the safety of cell application.

[0016] 3. hESC-MSCs in this invention OE-PGE-2 The proliferation potential of MSCs is stronger than that of MSCs. In this invention, they can be expanded to more than 50 generations and can differentiate into a certain number of seed cells before being expanded in large quantities, which greatly improves the cell reserve and overcomes the limitations of proliferation rate and senescence and apoptosis that occur during passage culture of currently used MSCs, which cannot cope with the problem of insufficient number of seed cells in sudden situations.

[0017] 4. hESC-MSCs in this invention OE-PGE-2 It utilizes gene editing technology to anchor and continuously express PGE-2 on its membrane, overcoming the low efficiency of currently used MSC gene editing, achieving the goal of precision treatment, and greatly improving the therapeutic effect of anti-liver fibrosis.

[0018] 5. The preparation method of this invention differentiates hESCs into MSCs (hESC-derived MSCs, hESC-MSCs) by adding growth factors such as EGF and bFGF, which serve as an important source of MSC seed cells and solve the problem of limiting the immunogenicity of embryonic stem cells.

[0019] 6. The preparation method of the present invention has the advantages of non-invasively obtaining seed cells and the seed cells having advantages such as editability, reproducibility, and good stability. Attached Figure Description

[0020] Figure 1hESC OE-PGE-2 The results of the establishment and identification are shown in the figure, where A is a diagram of the lentiviral vector with membrane-anchored PGE-2 overexpression; B is a diagram of hESC. OE-PGE-2 Identification; C and D are for in vitro gene expression assays (hESC). OE-PGE-2 Figure 1 shows the results of cell pluripotency experiments; Figures E and F show the results of in vivo teratoma formation experiments.

[0021] Figure 2 Flowchart for the differentiation of embryonic stem cells (hESCs) into mesenchymal stem cells (MSCs).

[0022] Figure 3 for hESC-MSCs OE-PGE-2 The characterization results are shown in the figure.

[0023] Figure 4 These are images showing the results of HE pathological staining, where A is the control group, B is the model group, C is the model group + primary MSCs, and D is the model group + hESC-MSCs. OE-PGE-2 .

[0024] Figure 5 The images show the results of Sirius red staining, where A is the control group, B is the model group, C is the model group + primary MSCs, and D is the model group + hESC-MSCs. OE-PGE-2 . Detailed Implementation

[0025] The present invention will be further described below with reference to specific embodiments, but this does not limit the scope of protection and application of the present invention.

[0026] I. Preparation of mesenchymal stem cells derived from embryonic stem cells expressing PGE-2 anchored on membrane Example 1

[0027] The preparation of mesenchymal stem cells derived from embryonic stem cells that express PGE-2 via membrane anchoring includes the following steps: 1. Preparation of membrane-anchored PGE-2 overexpressing hESC cell line (hESC OE-PGE-2 ).

[0028] (1) A membrane-anchored PGE-2 cloning vector was constructed using cloning technology and packaged into lentiviruses to obtain membrane-anchored lentiviruses overexpressing PGE-2, see [link to relevant documentation]. Figure 1 As shown in A; (2) hESCs were infected with lentiviruses that overexpressed PGE-2 and were then sorted by flow cytometry to obtain stably membrane-anchored hESCs. OE-PGE-2 Cell lines, see Figure 1 B. Flow cytometry results showed successful membrane expression of PGE-2, hESC. OE-PGE-2Even after differentiating into MSCs, they continue to express PGE-2; (3) For hESC OE-PGE-2 For identification, see Figure 1 C, D, E, and F indicate that membrane expression of PGE-2 does not affect the pluripotency of embryonic stem cells, thus proving that this operation did not affect their genomic stability.

[0029] 2.hESC OE-PGE-2 Cell differentiation into hESC-MSCs OE-PGE-2 Cells (see) Figure 2 (As shown).

[0030] hESC OE-PGE-2 Cells were differentiated in vitro to obtain hESC-MSCs OE-PGE-2 Cells consist of the following three stages: In the first stage, the feeder cells were removed and hESCs were performed in Matri-Gel-coated cell culture dishes. OE-PGE-2 Cell culture, removal of Matri-Gel coating, and induction of hESCs within 3 days OE-PGE-2 Cellular self-differentiation; In the second stage, trypsin was used to differentiate the hESCs. OE-PGE-2 Cells were digested into single cells and cultured using the following medium: RPMI-1640 (basal medium) + 20% KOSR (gene knockout serum replacement medium) + 10 ng / mL bFGF (basal fibroblast growth factor) + 20 ng / mL PDGFAB (platelet-derived growth factor AB) + 10 ng / mLEGF (epidermal growth factor). Mesenchymal stem cell-like cells appeared and proliferated on day 7. In the third stage, cells were passaged every other day to enrich mesenchymal stem cell-like cells. The highly enriched mesenchymal stem cell-like cells were then sorted by flow cytometry and labeled with CD24. - / CD105 + MSCs were sorted and purified, and then cultured as single clones in 96-well plates. MSCs were expanded within 14-21 days to establish hESC-MSCs. OE-PGE-2 After 30 days of differentiation, purification, and amplification, early hESC-MSCs were... OE-PGE-2 Identification and testing were conducted to demonstrate the characteristics of its mesenchymal stem cells (see [link]). Figure 3 The results showed that differentiated hESC-MSCs OE-PGE-2 It expresses surface markers of mesenchymal stem cells: CD90, CD105, CD29, and CD44; it does not express CD34, HLA-DR, CD45, or CD14; and it meets the identification criteria for mesenchymal stem cells.

[0031] II. Biological Research Example 2

[0032] tail vein injection of hESC-MSCs OE-PGE-2 Cells can effectively alleviate the progression of liver fibrosis in mice. 1. Experimental Materials 1.1 Laboratory Animals C57BL / 6 mice, SPF grade, 6-8 weeks old, male; purchased from Guangdong Provincial Experimental Animal Center, certificate number: No. 44007200042765.

[0033] 1.2. Therapeutic cells Primary MSCs (purchased from Cyagen, catalog number HUXMA-01001), and the cell product hESC-MSCs of this invention. OE -PGE-2 .

[0034] 1.3 Main Reagents MSCs culture medium (STEMCELL); sodium chloride (Sigma-Aldrich); phosphate buffer (PBS, Gibco); dimethyl sulfoxide (Gibco); Sirius red staining (Solepro, G1472).

[0035] 1.4 Main Instruments Clean bench, CO2 incubator (Thermo Scientific, USA); inverted microscope (Chongqing Aote Optical Instrument Co., Ltd.); low-speed centrifuge, high-speed centrifuge (Eppendorf, Germany); ChemiDoc TM Touch imaging system, electrophoresis apparatus (Bio-Rad, USA); real-time PCR instrument (Applied Biosystems, USA).

[0036] 2. Experimental Methods 2.1 Establishment, grouping, and intervention of a liver fibrosis model Thirty-two male C57BL / 6 mice aged 6–8 weeks were used. Twenty-four mice were randomly selected for bile duct ligation (BDL) to establish a liver fibrosis model, while the other eight mice served as a blank control group, undergoing only laparotomy without surgery. One week later, the 24 surgically treated mice were randomly divided into a model group, a primary MSCs treatment group, and an hESC-MSCs group. OE-PGE-2 Treatment groups, 8 mice per group; primary MSCs treatment group and hESC-MSCs treatment group. OE-PGE-2 The treatment group received a tail vein injection. Cells, control group and model group were injected with sterile 0.9% sodium chloride via tail vein, once every 1 day, 3 times a week, and mice were harvested 7 days after the last intervention.

[0037] 2.2 Hematoxylin-eosin (HE) staining of liver tissue After sampling, liver tissue was fixed with 10% formic acid, routinely dehydrated, embedded in paraffin, and prepared into paraffin blocks. Paraffin samples were sectioned using a paraffin microtome to a thickness of 3.5 μm, with serial sections. The staining procedure was as follows: drying the slide for 20 minutes, xylene twice for 10 minutes, anhydrous ethanol twice for 2 minutes, 95% ethanol for 1 minute, 80% ethanol for 1 minute, 70% ethanol for 1 minute, washing with water for 1 minute, hematoxylin for 8 minutes, hematoxylin for 10 minutes, washing with water twice for 1 minute, 0.5% hydrochloric acid alcohol for 10 seconds, washing with water for 10 minutes, eosin for 2 minutes, washing with water for 1 minute, 80%, 85%, and 90% ethanol for 5 seconds each, 95% ethanol for 1 minute, anhydrous ethanol twice for 2 minutes, anhydrous ethanol for 3 minutes, xylene twice for 2 minutes. After staining, the slides were mounted directly with neutral resin and examined under a microscope.

[0038] 2.3 Sirius Red staining of liver tissue After sampling, the liver tissue was stained with Sirius red. For specific operating procedures, please refer to the instruction manual.

[0039] 2.4 Statistical Methods Image data were processed and analyzed using ImageJ software, while other data were statistically processed and analyzed using SPSS 17.0 software. P < 0.05 was considered statistically significant.

[0040] 3. Experimental Results 3.1 Hematoxylin-eosin (HE) staining of liver tissue See Figure 4 HE pathological staining showed that extracellular matrix (ECM) formation and extensive cell death were observed in mice in the BDL liver fibrosis model group, while primary MSCs and hESC-MSCs showed significantly better results. OE-PGE-2 After treatment, ECM deposition and fibrosis were alleviated, and hESC-MSCs OE-PGE-2 The effect is more significant, indicating that hESC-MSCs OE-PGE-2 It has a better therapeutic effect on liver damage.

[0041] 3.2 Sirius Red Staining of Liver Tissue See results Figure 5Sirius red staining (a specific staining for fibrosis in the extracellular matrix) showed tightly packed type I collagen fibers with strong birefringence, appearing as red fibers. Simultaneously, a large amount of type I collagen fiber deposition was observed in mice in the BDL liver fibrosis model group, while primary MSCs and hESC-MSCs... OE-PGE-2 After treatment, type I collagen fiber deposition and fibrosis were alleviated, and hESC-MSCs showed improvement. OE-PGE-2 The treatment effect is more significant.

Claims

1. A method for preparing mesenchymal stem cells derived from embryonic stem cells expressing PGE-2 via membrane anchoring, characterized in that, Includes the following steps: (1) Preparation of membrane-anchored PGE-2 overexpressing hESC cell line hESC OE-PGE-2 ; (2) hESC OE-PGE-2 Cell differentiation into hESC-MSCs OE-PGE-2 cell; Step (1) includes the following steps: S1. A membrane-anchored PGE-2 cloning vector was constructed using cloning technology and packaged into a lentivirus to obtain a membrane-anchored lentivirus overexpressing PGE-2; S2. Infection of embryonic stem cells with lentiviruses that overexpress PGE-2 via membrane anchoring; S3. Stable membrane-anchored hESCs were obtained by flow cytometry sorting. OE-PGE-2 Cell lines; Step (2) includes the following three stages: In the first stage, the feeder cells were removed and hESC was performed in Matri-Gel-coated cell culture dishes. OE-PGE-2 Cell culture, removing the Matri-Gel coating condition in the culture system, and inducing hESCs OE-PGE-2 Cellular self-differentiation; In the second stage, trypsin was used to differentiate the hESCs. OE-PGE-2 Cells are digested into single cells and cultured, which promotes the proliferation of mesenchymal stem cell-like cells; In the third stage, cells were passaged every other day to enrich mesenchymal stem cell-like cells. The highly enriched mesenchymal stem cell-like cells were then sorted by flow cytometry and labeled with CD24. - / CD105 + MSCs were sorted and purified, and then cultured as single clones in 96-well plates to expand MSCs and establish hESC-MSCs. OE-PGE-2 Tie.

2. The method for preparing mesenchymal stem cells derived from membrane-anchored PGE-2-expressing embryonic stem cells according to claim 1, characterized in that, Step (2) The induction time in the first stage is 3 days.

3. The method for preparing mesenchymal stem cells derived from membrane-anchored PGE-2-expressing embryonic stem cells according to claim 1, characterized in that, The culture and proliferation time in the second stage of step (2) is 4 days.

4. The method for preparing mesenchymal stem cells derived from membrane-anchored embryonic stem cells expressing PGE-2 according to claim 1, characterized in that, Step (2) The components of the culture medium used in the second stage of cultivation include: RPMI-1640, 20% KOSR, 10 ng / mL bFGF, 20 ng / mL PDGFAB, and 10 ng / mL EGF.

5. The method for preparing mesenchymal stem cells derived from membrane-anchored PGE-2-expressing embryonic stem cells according to claim 1, characterized in that, Step (2) The third stage of cultivation takes 7 to 14 days.

6. An application of mesenchymal stem cells derived from embryonic stem cells that express PGE-2 on a membrane, characterized in that, Mesenchymal stem cells are prepared using any one of the methods described in claims 1-5, and the application is in the preparation of anti-hepatic fibrosis drugs.

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