Method for preparing erythropoietin-producing cells and use thereof

Abstract

This invention discloses a method for preparing erythropoietin-producing cells and their uses. The method induces pluripotent stem cells to differentiate into erythropoietin-producing cells, which then secrete erythropoietin. This method not only eliminates the need for a hypoxic environment to produce erythropoietin in the cells, but also is simple to operate, requires few reagents, and has a short differentiation cycle, effectively solving the technical problem of low erythropoietin production. Furthermore, the erythropoietin-producing cells of this invention can also be used as cell models for scientific research, including but not limited to: research on the protective effect and molecular mechanism of erythropoietin-mediated renal ischemia, research on the pathophysiological mechanism of anemia, and research on the protective mechanism of myocardial ischemia.

Description

Technical Field

[0001] This invention relates to the field of cell culture technology, specifically to a method for preparing erythropoietin-producing cells and their applications. Background Technology

[0002] Erythropoietin (EPO) is a glycoprotein hormone with multiple physiological functions, its core function being to promote red blood cell production. Currently, EPO is widely used clinically to treat various types of anemia, especially renal anemia. Marketed drugs include dapoxetine alpha (trade name: NESP / Aranesp), methoxy polyethylene glycol erythropoietin (trade name: Mircera), and pemoxatide (trade name: Saint Laurent). EPO can be produced by the human body, primarily from the kidneys (pericytes, interstitial cells, and distal convoluted tubule cells), with a small amount produced by the liver. When the body's EPO levels are low, exogenous EPO (the aforementioned drugs) can be injected or supplemented. Currently, EPO is mainly obtained through gene recombination technology, while the yield obtained through pluripotent stem cell (PSC) culture technology is too low, and the culture process involves many reagents, is cumbersome, and costly. Therefore, the industrial application of this preparation method is limited. Summary of the Invention

[0003] This invention aims to at least solve one of the aforementioned technical problems existing in the prior art. This invention provides a method for preparing erythropoietin-producing cells and its uses. The method enables induced pluripotent stem cells (iPSCs) to differentiate into erythropoietin-producing cells and secrete erythropoietin. The method of this invention not only eliminates the need to provide a hypoxic environment for the erythropoietin-producing cells to produce erythropoietin, but also is simple to operate, requires few reagents, and has a short differentiation cycle, effectively solving the technical problem of low erythropoietin production.

[0004] To achieve the above objectives, the present invention adopts the following technical solution: In a first aspect, the present invention provides a method for preparing erythropoietin-producing cells, the method using pluripotent stem cells as the starting point, and comprising the following steps: 1) Pluripotent stem cells were cultured in the first culture medium for 2-6 days, which contained GSK-3 inhibitors; 2) The cells obtained in step 1) are induced to differentiate into erythropoietin-producing cells using a second culture medium containing a TGF-β receptor kinase inhibitor, a RAR / RXR nuclear receptor agonist and fibroblast growth factor 2 (FGF2).

[0005] In some embodiments of the present invention, the pluripotent stem cells are human induced pluripotent stem cells, and the human induced pluripotent stem cells are preferably induced pluripotent stem cells derived from peripheral blood mononuclear cells.

[0006] In some embodiments of the present invention, the first culture medium is cultured for 2.5 to 3.5 days.

[0007] In some embodiments of the present invention, the induction time of the second culture medium is at least 2 days.

[0008] In some embodiments of the present invention, the GSK-3 inhibitor in the first culture medium is one or a combination of CHIR99021, CHIR98014, and CHIR98023.

[0009] In some embodiments of the present invention, the TGF-β receptor kinase inhibitor in the second culture medium includes SB431542.

[0010] In some embodiments of the present invention, the RAR / RXR nuclear receptor agonist in the second culture medium includes retinoic acid (RA).

[0011] In some embodiments of the present invention, the concentration of the GSK-3 inhibitor in the first culture medium is 2-12 μM; for example, the concentration of the GSK-3 inhibitor in the first culture medium may be 2 μM, 3 μM, 4 μM, 5 μM, 6 μM, 7 μM, 8 μM, 9 μM, 10 μM, 11 μM, or 12 μM.

[0012] In some embodiments of the present invention, the concentration of the TGF-β receptor kinase inhibitor in the second culture medium is 1-20 μM; for example, the concentration of the TGF-β receptor kinase inhibitor in the second culture medium may be 1 μM, 2 μM, 3 μM, 4 μM, 5 μM, 6 μM, 7 μM, 8 μM, 9 μM, 10 μM, 11 μM, 12 μM, 13 μM, 14 μM, 15 μM, 16 μM, 17 μM, 18 μM, 19 μM, or 20 μM.

[0013] In some embodiments of the present invention, the concentration of the RAR / RXR nuclear receptor agonist in the second culture medium is 0.1-5 μM; for example, the concentration of the RAR / RXR nuclear receptor agonist in the second culture medium may be 0.1 μM, 0.2 μM, 0.3 μM, 0.4 μM, 0.5 μM, 0.6 μM, 0.7 μM, 0.8 μM, 0.9 μM, 1.0 μM, 2 μM, 3 μM, 4 μM, or 5 μM.

[0014] In some embodiments of the present invention, the concentration of FGF2 in the second culture medium is 10-200 ng / mL; for example, the concentration of FGF2 in the second culture medium can be 10 ng / mL, 20 ng / mL, 30 ng / mL, 40 ng / mL, 50 ng / mL, 60 ng / mL, 70 ng / mL, 80 ng / mL, 90 ng / mL, 100 ng / mL, 110 ng / mL, 120 ng / mL, 130 ng / mL, 140 ng / mL, 150 ng / mL, 160 ng / mL, 170 ng / mL, 180 ng / mL, 190 ng / mL, or 200 ng / mL.

[0015] In some embodiments of the present invention, the first culture medium comprises RPMI 1640.

[0016] In some embodiments of the present invention, the second culture medium comprises RPMI 1640.

[0017] In some embodiments of the present invention, the first culture medium and the second culture medium further include one or two of serum substitutes and MEM non-essential amino acid solutions (NEAA).

[0018] In some embodiments of the present invention, the working concentration of the serum substitute is 5-20 v / v.

[0019] In some embodiments of the present invention, the serum substitute is KnockOut™ Serum Replacement (KOSR).

[0020] In some embodiments of the present invention, the working concentration of the NEAA is 0.1-10 v / v.

[0021] In some embodiments of the present invention, the cell culture in step 1) is carried out in an environment of 36-38°C and 4.0-6.0% CO2; the cell induction in step 2) is carried out in an environment of 36-38°C and 4.0-6.0% CO2.

[0022] A second aspect of the present invention provides the use of erythropoietin-producing cells prepared according to the above-described preparation method in the preparation of erythropoietin.

[0023] The beneficial effects of this invention are: This invention provides a method for preparing erythropoietin-producing cells and their applications. The method induces pluripotent stem cells to differentiate into erythropoietin-producing cells, which then secrete erythropoietin. This method not only eliminates the need for a hypoxic environment in the erythropoietin-producing cells to produce erythropoietin, but also is simple to operate, requires few reagents, and has a short differentiation cycle, effectively solving the technical problem of low erythropoietin production in mammalian cells under conventional culture conditions. Furthermore, the erythropoietin-producing cells of this invention can also be used as cell models for scientific research, including but not limited to: research on the protective effect and molecular mechanism of erythropoietin-mediated renal ischemia, research on the pathophysiological mechanism of anemia, and research on the protective mechanism of myocardial ischemia. Attached Figure Description

[0024] Figure 1 The EPO yields of Examples 1-2 and Comparative Examples 1-3 are shown.

[0025] Figure 2 The relative expression levels of the OSR1 gene, a marker of renal interstitial cells, are shown in Example 1 and Comparative Examples 1-3.

[0026] Figure 3 The relative expression levels of FOXD1, CD140b, and CD73 genes are shown in Example 1 and Comparative Examples 1-3.

[0027] Figure 4 The results of cell immunofluorescence in Example 1 are shown, in which... Figure 4 (a) shows cells from Example 1 co-stained with EPO (red fluorescence) and CD73 (green fluorescence). Figure 4 (b) shows cells from Example 1 co-stained with EPO (red fluorescence) and CD140b (green fluorescence).

[0028] Figure 5 The flow cytometry results of EPO and CD73 obtained from erythropoietin-producing cells in Example 1 and human induced pluripotent stem cells (control group) are shown. Detailed Implementation

[0029] The present invention will be further described in detail below through specific embodiments. Unless otherwise specified, the raw materials, reagents, or apparatus used in the embodiments and comparative examples are all available from conventional commercial sources or can be obtained by existing technical methods. Unless otherwise specified, the test or experimental methods are conventional methods in the art.

[0030] Example 1 This embodiment provides a method for preparing erythropoietin-producing cells, the specific steps of which are as follows: Step S1: Add 3 mL of Matrigel matrix gel to a T25 culture dish, spread the matrix gel evenly, and incubate at 37°C for 2 hours. Then inoculate with 1×10⁻⁶ cells / mL. 6 Personal induced pluripotent stem cells were cultured in a matrix gel containing 10 v / v% KOSR, 1 v / v% NEAA and 8 μM CHIR99021 for 3 days.

[0031] Step S2: Change the medium to RPMI 1640 containing 10 v / v% KOSR, 1 v / v% NEAA, 10 μM SB 431542, 0.5 μM retinoic acid and 100 ng / mL FGF2 and culture the cells for 2 days to obtain erythropoietin-producing cells and collect the supernatant for later use.

[0032] Cell culture environment for steps S1 and S2: 37.0℃, 5.0% CO2 carbon dioxide incubator, ambient temperature: 18-26℃, ambient humidity: 45-65%.

[0033] Example 2 This embodiment provides a method for preparing erythropoietin-producing cells, the specific steps of which are as follows: Step S1: Add 3 mL of Matrigel matrix gel to a T25 culture dish, spread the matrix gel evenly, and incubate at 37°C for 2 hours. Then inoculate with 1×10⁻⁶ cells / mL. 6 Personal induced pluripotent stem cells were cultured in a matrix gel containing RPMI 1640 medium with 10 v / v% KOSR, 1 v / v% NEAA and 5 μM CHIR99021 for 3 days.

[0034] Step S2: Change the medium to RPMI 1640 containing 10 v / v% KOSR, 1 v / v% NEAA, 5 μM SB 431542, 0.1 μM retinoic acid and 10 ng / mL FGF2 and culture the cells for 2 days to obtain erythropoietin-producing cells and collect the supernatant for later use.

[0035] Cell culture environment for steps S1 and S2: 37.0℃, 5.0% CO2 carbon dioxide incubator, ambient temperature: 18-26℃, ambient humidity: 45-65%.

[0036] Comparative Example 1 This comparative example provides a method for preparing erythropoietin-producing cells, which differs from Example 1 in that step S2 lacks SB 431542. The specific steps are as follows: Step S1: Add 3 mL of Matrigel matrix gel to a T25 culture dish, spread the matrix gel evenly, and incubate at 37°C for 2 hours. Then inoculate with 1×10⁻⁶ cells / mL. 6 Personal induced pluripotent stem cells were cultured in a matrix gel containing 10 v / v% KOSR, 1 v / v% NEAA and 8 μM CHIR99021 for 3 days.

[0037] Step S2: Change the medium to RPMI1640 containing 10 v / v% KOSR, 1 v / v% NEAA, 0.5 μM retinoic acid and 100 ng / mL FGF2 and culture the cells for 2 days to obtain erythropoietin-producing cells, and collect the supernatant for later use.

[0038] Comparative Example 2 This comparative example provides a method for preparing erythropoietin-producing cells, which differs from Example 1 in that step S2 lacks retinoic acid. The specific steps are as follows: Step S1: Add 3 mL of Matrigel matrix gel to a T25 culture dish, spread the matrix gel evenly, and incubate at 37°C for 2 hours. Then inoculate with 1×10⁻⁶ cells / mL. 6 Personal induced pluripotent stem cells were cultured in a matrix gel containing 10 v / v% KOSR, 1 v / v% NEAA and 8 μM CHIR99021 for 3 days.

[0039] Step S2: Change the medium to RPMI 1640 containing 10 v / v% KOSR, 1 v / v% NEAA, 10 μM SB 431542 and 100 ng / mL FGF2 and culture the cells for 2 days to obtain erythropoietin-producing cells and collect the supernatant for later use.

[0040] Comparative Example 3 This comparative example provides a method for preparing erythropoietin-producing cells, which differs from Example 1 in that step S2 lacks FGF2. The specific steps are as follows: Step S1: Add 3 mL of Matrigel matrix gel to a T25 culture dish, spread the matrix gel evenly, and incubate at 37°C for 2 hours. Then inoculate with 1×10⁻⁶ cells / mL. 6 Personal induced pluripotent stem cells were cultured in a matrix gel containing 10 v / v% KOSR, 1 v / v% NEAA and 8 μM CHIR99021 for 3 days.

[0041] Step S2: Change the culture medium to RPMI 1640 containing 10 v / v% KOSR, 1 v / v% NEAA, 10 μM SB 431542 and 0.5 μM retinoic acid and culture the cells for 2 days to obtain erythropoietin-producing cells, and collect the supernatant for later use.

[0042] Detection Example 1 The EPO content of the supernatants collected in Examples 1, 2 and Comparative Examples 1-3 was determined according to the procedures described in the Beyotime Human EPO ELISA Kit instruction manual.

[0043] Result: As Figure 1 As shown, the EPO content in the culture supernatant of Examples 1 and 2 was significantly higher than that in Comparative Examples 1-3. Therefore, the method in Example 1 (using SB 431542, retinoic acid and FGF2 in combination) can better induce pluripotent stem cells to differentiate into erythropoietin-producing cells and secrete erythropoietin.

[0044] Detection Example 2 RNA was extracted from cells collected in step S1 of Example 1, and the relative expression level of the OSR1 gene, a marker of renal interstitial cells, was detected using the Beyotime Probe One-Step qRT-PCR Kit (the control group was human induced pluripotent stem cells, and the internal reference was the GAPDH gene). The primer sequences used to detect OSR1 and the internal reference GAPDH gene are shown in Table 1 below.

[0045] Table 1: Primer sequences

[0046] Results: OSR1 is a key marker gene in early kidney development, expressed in the mesenchyme and metanephric mesenchyme. The metanephric mesenchyme can differentiate into renal interstitial progenitor cells expressing FOXD1, which are the source of erythropoietin-producing cells. Figure 2 As shown, the relative expression level of the OSR1 gene, a marker of renal interstitial progenitor cells, in step S1 of Example 1 was significantly higher than that in comparative examples 1-3, reflecting that the human induced pluripotent stem cells of Example 1 differentiated into interstitial mesoderm or metarenal mesenchymal cells in step S1.

[0047] Detection Example 3 Erythropoietin-producing cells from Example 1, Comparative Example 1, Comparative Example 2, and Comparative Example 3 (step S2) were collected, and RNA was extracted. The expression levels of FOXD1, CD140b, and CD73 genes were detected using the Beyotime Probe One-Step qRT-PCR Kit (the control group was human induced pluripotent stem cells, and the internal reference was the GAPDH gene). The primer sequences used for the detection are shown in Table 2 below.

[0048] Table 2: Primer sequences

[0049] Result: As Figure 3 As shown, the cells in step S2 of Example 1 expressed the renal interstitial progenitor cell marker gene FOXD1, as well as the erythropoietin production marker genes CD140b and CD73, and their relative expression levels were significantly higher than those in comparative examples 1-3.

[0050] Detection Example 4 Cells from Example 1 were collected, transferred to cell slides, and subjected to immunofluorescence experiments, as follows: (1) Fixation: Discard the culture medium, add 500 μL of 1×PBS to wash 3 times, add an appropriate amount of 4% paraformaldehyde tissue fixative (200 μL per well in a 24-well plate), let stand for 15 min; discard the fixative, add 500 μL of 1×PBS, discard after 5 min, and repeat 3 times.

[0051] (2) Permeation: Add an appropriate amount of 0.1% PBS-Triton X-100 permeation solution to each well (24-well plate: 200 μL per well) and let stand for 15 min.

[0052] (3) Blocking: Discard the permeation solution, add 500 μL of 1×PBS, discard after 5 min, repeat 3 times, then add an appropriate amount of 5% goat serum blocking solution to each well (24-well plate: 200 μL per well), and incubate at room temperature for 1 h.

[0053] (4) Primary antibody incubation: Discard the blocking solution, add an appropriate amount of the corresponding primary antibody working solution to each well (200 μL per well for 24-well plate), and incubate the plate at 4°C overnight or at room temperature for 2 hours. The primary antibody information used is shown in Table 3 below: Table 3: Primary Antibody Information

[0054] (5) Secondary antibody incubation: Discard the primary antibody, add 500 μL of 1×PBS, discard after 5 min, and repeat the washing 3 times; add an appropriate amount of the corresponding secondary antibody working solution to each well (24-well plate: 200 μL per well), wrap the culture plate with tin foil, and let it stand at room temperature for 1 h.

[0055] (6) Washing: Discard the secondary antibody, add 500 μL of 1×PBS, discard after 5 min, and repeat the washing 3 times.

[0056] (7) Stain the nuclei with DAPI (1 μg / mL) for 5 minutes, wash twice with PBS, and then mount the slide.

[0057] Result: As Figure 4 As shown in (a) and (b), the immunofluorescence experiment results show that EPO and CD73 are significantly expressed in the cells of Example 1. Therefore, human induced pluripotent stem cells successfully differentiated into erythropoietin-producing cells, thereby secreting erythropoietin.

[0058] Case 5 Cells from Example 1 were collected for flow cytometry analysis, and the steps are as follows: Sample processing: Take 4 × 10⁻⁶ cells from Example 1 and human induced pluripotent stem cell suspensions respectively. 6 ~5×10 6 (1 cell) were placed into different EP tubes, centrifuged at 500g for 5 minutes, and the supernatant was discarded.

[0059] Antibody incubation: Add 5 μL of the corresponding antibody (EPO-FITC, CD73-APC) to the EP tubes respectively, and incubate at room temperature in the dark for 30 min. The antibody information is shown in Table 4 below.

[0060] Table 4: Antibody Information

[0061] Washing: Add 1 mL of 1X washing solution to each EP tube and mix thoroughly. Centrifuge at 500g for 5 min and discard the supernatant. Repeat the washing once.

[0062] Pre-processing: Add 300 μL of DPBS to each EP tube for resuspending, ready for instrumental testing.

[0063] For on-machine testing, the fluorescence channels were selected as 488nm and 525nm.

[0064] Result: As Figure 5 As shown, flow cytometry results indicate that EPO and CD73 are significantly expressed, thus human induced pluripotent stem cells successfully differentiate into erythropoietin-producing cells, thereby secreting erythropoietin.

[0065] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A method for preparing erythropoietin-producing cells, characterized in that, Includes the following steps: 1) Pluripotent stem cells were cultured in the first culture medium for 2-6 days, which contained GSK-3 inhibitors; 2) The cells obtained in step 1) are induced to differentiate into erythropoietin-producing cells using a second culture medium containing a TGF-β receptor kinase inhibitor, a RAR / RXR nuclear receptor agonist and fibroblast growth factor 2.

2. The preparation method according to claim 1, characterized in that, The GSK-3 inhibitor in the first culture medium is one or a combination of CHIR99021, CHIR98014, and CHIR98023.

3. The preparation method according to claim 1, characterized in that, The TGF-β receptor kinase inhibitors in the second culture medium include SB 431542.

4. The preparation method according to claim 1, characterized in that, The RAR / RXR nuclear receptor agonist in the second culture medium includes retinoic acid.

5. The preparation method according to claim 1, characterized in that, The concentration of the GSK-3 inhibitor in the first culture medium was 2-12 μM.

6. The preparation method according to claim 1, characterized in that, The concentration of the TGF-β receptor kinase inhibitor in the second culture medium was 1-20 μM.

7. The preparation method according to claim 1, characterized in that, The concentration of the RAR / RXR nuclear receptor agonist in the second culture medium was 0.1-5 μM.

8. The preparation method according to claim 1, characterized in that, The concentration of FGF2 in the second culture medium was 10-200 ng / mL.

9. The preparation method according to claim 1, characterized in that, The first culture medium and the second culture medium also include one or two of serum substitutes and MEM non-essential amino acid solutions.

10. Use of the erythropoietin-producing cells prepared by the method according to any one of claims 1-9 in the preparation of erythropoietin.

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