CN113373114A - Culture medium and method for improving efficiency of differentiation of pluripotent stem cells into hematopoietic stem cells - Google Patents

Culture medium and method for improving efficiency of differentiation of pluripotent stem cells into hematopoietic stem cells Download PDF

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CN113373114A
CN113373114A CN202110652831.7A CN202110652831A CN113373114A CN 113373114 A CN113373114 A CN 113373114A CN 202110652831 A CN202110652831 A CN 202110652831A CN 113373114 A CN113373114 A CN 113373114A
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culture medium
stem cells
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曹哲厚
吴立前
韦俊
马悦悦
王振坤
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Hangzhou Yuansheng Biotechnology Co ltd
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Abstract

The invention discloses a culture medium and a method for improving the efficiency of differentiating pluripotent stem cells into hematopoietic stem cells, and the culture medium comprises a first-stage culture medium, a second-stage culture medium and a third-stage culture medium; the first stage culture medium comprises E8 complete culture medium, SCF, rhG-CSF, VEGF, PDGF; the second stage culture medium comprises E6 basal medium, CHIR99021, VEGF, SCF, IL-3 and CSF; the third stage culture medium comprises a Stemline II basal culture medium, SCF, HGH, Flt3L, EPO, EGF and a novel pyrimidine indole compound; the present invention greatly improves the efficiency of differentiation of pluripotent stem cells into hematopoietic stem cells by appropriately using a culture medium in which a novel pyrimidine indole compound is combined with the above three specific components and concentrations to induce differentiation of pluripotent stem cells into hematopoietic stem cells, so that a large number of in vitro cultured hematopoietic stem cells can be obtained in a short period of time.

Description

Culture medium and method for improving efficiency of differentiation of pluripotent stem cells into hematopoietic stem cells
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a culture medium and a method for improving the efficiency of differentiation of pluripotent stem cells into hematopoietic stem cells.
Background
Hematopoietic Stem Cells (HSCs) are a small population of the most primitive Hematopoietic cells with high self-replicating and multipotent differentiation potential. That is, hematopoietic stem cells have two important characteristics: first, a high degree of self-renewal or self-replication ability; second, all types of blood cells can be generated differentially. Genetically, hematopoietic stem cells belong to one of the adult stem cells. Hematopoietic stem cells are also a pluripotent stem cell because they differentiate into at least 12 types of blood cells.
The application of the hematopoietic stem cells mainly comprises hematopoietic stem cell transplantation and the application of the hematopoietic stem cells as target cells of gene therapy, and the hematopoietic stem cells have the advantages of easy material taking, easy in-vitro culture, easy in-vivo transplantation into a patient, high survival and self-renewal capacity and the like, so the hematopoietic stem cells are one of ideal target cells of the gene therapy. Exogenous genes are transferred into hematopoietic stem cells collected from a patient, and the hematopoietic stem cells are returned to the patient, where they are expressed in vivo to treat diseases. To date, hematopoietic stem cells have been used for gene therapy for Adenosine Deaminase (ADA) deficiency, Gaucher disease (Gaucher disease), HIV infection, and cancer. However, hematopoietic stem cells are a great problem in quantity. First hematopoietic stem cells are quiescent and rarely divide and the source of embryonic stem cells is now limited; the operation process of the human embryo for somatic cell cloning is complicated, the cost is too high, and the ethics of the human embryo is not generally accepted by people; the induction, proliferation and directional differentiation of stem cells in vitro have certain technical difficulties, and the failure to culture hematopoietic stem cells in vitro can delay and hinder the development process of the new therapy.
In the traditional method, the efficiency of differentiating the pluripotent stem cells into hematopoietic stem cells is low, and serum is usually added into an induced differentiation culture medium of the traditional method to promote cell proliferation, and the pluripotent stem cells are co-cultured with different stromal cells, such as OP9 cells and the like, and are directly directionally induced and differentiated into hematopoietic cells; however, the use of serum in this conventional method increases the source of contamination, and the use of OP9 cells poses unknown risks and is of low safety.
Disclosure of Invention
The invention provides a culture medium and a method for improving the efficiency of differentiating pluripotent stem cells into hematopoietic stem cells in order to overcome the defects of the prior art.
In order to achieve the purpose, the invention adopts the following technical scheme: a culture medium for inducing differentiation of pluripotent stem cells into hematopoietic stem cells comprises a first-stage culture medium, a second-stage culture medium and a third-stage culture medium;
the first stage culture medium comprises an E8 complete culture medium, SCF, rhG-CSF, VEGF and PDGF, and the final concentration ratio of the SCF, the rhG-CSF, the VEGF and the PDGF in the first stage culture medium is (1.0-2.0) to (0.25-0.5) to (1.0-2.0);
the second stage culture medium comprises an E6 basal culture medium, CHIR99021, VEGF, SCF, IL-3 and CSF, and the final concentration ratio of the CHIR99021, the VEGF, the SCF, the IL-3 and the CSF in the second stage culture medium is (0.25-1.0): (1.0-2.0): (0.5-2.0);
the third stage culture medium comprises a Stemline II basal culture medium, SCF, HGH, Flt3L, EPO, EGF and novel pyrimidine indole compounds, and the final concentration ratio of the SCF, the HGH, the Flt3L, the EPO, the EGF and the novel pyrimidine indole compounds in the third stage culture medium is (0.5-2.0): (0.2-0.4): (0.5-2.0): (1.0-2.0): (0.2-0.4).
Optionally, the novel pyrimidine indole compounds include one or both of UM171 and UM 729.
Optionally, the final concentration of SCF in the first stage culture medium is 50-100ng/mL, the final concentration of rhG-CSF is 50-100ng/mL, the final concentration of VEGF is 10-50mM/mL, and the final concentration of PDGF is 50-100 ng/mL; the final concentration of CHIR99021 in the second stage culture medium is 1-5 mu M, VEGF and is 50-100ng/mL, the final concentration of SCF is 20-80ng/mL, the final concentration of IL-3 is 20-80ng/mL, and the final concentration of CSF is 20-80 ng/mL; the final concentration of SCF in the third stage culture medium is 20-80ng/mL, the final concentration of HGH is 10-100ng/mL, the final concentration of Flt3L is 20-80ng/mL, the final concentration of EPO is 20-80ng/mL, the final concentration of EGF is 20-80ng/mL, and the final concentration of the novel pyrimidine indole compound is 10-50 mu M.
Optionally, the final concentration ratio of SCF, rhG-CSF, VEGF, PDGF in the first stage culture medium is 1.0: 0.25: 1.0; the final concentration ratio of CHIR99021, VEGF, SCF, IL-3 and CSF in the second-stage culture medium is 0.25: 1.0: 0.625; the final concentration ratio of SCF, HGH, Flt3L, EPO, EGF and the novel pyrimidine indole compound in the third stage culture medium is 1.0: 0.5: 1.0: 2.0: 0.2.
Optionally, the final concentration of SCF in the first stage culture medium is 80ng/mL, the final concentration of rhG-CSF is 80ng/mL, the final concentration of VEGF is 20ng/mL, and the final concentration of PDGF is 80 ng/mL; the final concentration of CHIR99021 in the second-stage culture medium is 80ng/mL for a final concentration of 2 mu M, VEGF, 80ng/mL for a final concentration of SCF, 50ng/mL for a final concentration of IL-3 and 50ng/mL for a final concentration of CSF; the final concentration of VEGF in the third stage culture medium is 40ng/mL, the final concentration of SCF is 50ng/mL, the final concentration of HGH is 10ng/mL, the final concentration of Flt3L is 20ng/mL, the final concentration of EPO is 40ng/mL, the final concentration of EGF is 40ng/mL, and the final concentration of the novel pyrimidine indole compound is 35 mu M.
The invention also discloses a method for improving the efficiency of differentiating the pluripotent stem cells into the hematopoietic stem cells, which comprises the following steps:
s1: placing pluripotent stem cells in an Aggrewell plate, and performing first-stage induced differentiation into embryoid bodies in the first-stage medium according to any one of claims 1 to 5;
s2: placing the embryoid body in a second-stage culture medium of any one of claims 1 to 5 for a second-stage induced differentiation;
s3: placing the cell mixture after induced differentiation of S2 in the third-stage medium according to any one of claims 1 to 5, and performing third-stage induced differentiation.
Optionally, the first stage of culturing the pluripotent stem cells inoculated on an Aggrewell plate for embryoid body differentiation is recorded as day 0, the induced differentiation of the first stage starts from day 0 to day 4, the induced differentiation of the second stage starts from day 4 to day 6, and the induced differentiation of the third stage starts from day 8 to day 12.
Optionally, on the 0 th day to the 4 th day of the induced differentiation of the first stage, the first stage differentiation medium is used in the inoculation period, and the liquid is not changed; transferring the embryoid bodies to a T75 culture bottle for culture to the 6 th day on the 4 th day of the second stage of induced differentiation, and adding a second stage of differentiation culture medium during the transfer period without changing the medium; on the 6 th day of the third stage of induced differentiation, the medium in the T75 flask was replaced with fresh medium for the third stage, and half every 3 days from the 6 th day of the third stage of induced differentiation was replaced with fresh medium for the third stage. During the third stage culture period, the culture medium is replaced by fresh third stage culture medium for half a day every 3 days, so that sufficient cell factors and nutrients are provided for cell differentiation, rapid cell differentiation is promoted, and the yield of hematopoietic stem cells can be greatly increased by timely replacing liquid.
Optionally, the number of pluripotent stem cells seeded in the Aggrewell plate is 30 ten thousand cells per well.
Optionally, the pluripotent stem cells include one or both of induced pluripotent stem cells and embryonic stem cells.
In conclusion, the invention has the following beneficial effects:
1. the invention greatly improves the efficiency of the differentiation of the pluripotent stem cells into the hematopoietic stem cells by properly using a culture medium which uses a novel pyrimidine indole compound to be matched with the three specific components and the concentrations to induce the differentiation of the pluripotent stem cells into the hematopoietic stem cells, so that a large amount of in vitro cultured hematopoietic stem cells can be obtained in a short time;
2. the pure cell factor method is used for preparing the culture medium, so that the use of serum is avoided, and a pollution source is reduced so as to avoid pollution; the invention avoids using other cell lines as the feeding induction conditions, so that the differentiated cells are safer and the unknown risk is reduced.
Drawings
FIG. 1 is a graph showing the results of flow-assay of the CD34+ positivity of example 1 and its control group.
FIG. 2 is a graph showing the results of flow-assay of the CD34+ positivity of example 2 and its control group.
Fig. 3 is a graph showing the results of flow-detecting the CD34+ positive rate of the negative group.
FIG. 4 is a bar graph of the positive rates of example 1 and its comparative group, and example 2 and its comparative group.
FIG. 5 is a histogram of the fold expansion of differentiation for example 1 and its comparative group, example 2 and its comparative group.
Detailed Description
In order to make the technical solutions of the present invention better understood, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention.
The cells adopted by the invention are purchased from the institute of biotechnology, Beijing Beinanna Chuanglian union.
The ultimate goal of studying the proliferation and differentiation mechanisms of stem cells is to apply the cells to treat diseases. The invention provides a culture medium and a method for improving the efficiency of differentiating pluripotent stem cells into hematopoietic stem cells.
Example 1
This example provides a medium and method for inducing differentiation of embryonic stem cells (ES) into hematopoietic stem cells by cytokine method.
The culture medium used in this example includes a first-stage culture medium, a second-stage culture medium, and a third-stage culture medium;
in the first stage culture medium, the final concentration ratio of SCF, rhG-CSF, VEGF and PDGF is 1.0: 0.25: 1.0, and the specific formula is as follows: to the E8 complete medium were added Stem Cell Factor (SCF) at a final concentration of 80ng/mL, recombinant human granulocyte colony-stimulating factor (rhG-CSF) at a final concentration of 80ng/mL, VEGF at a final concentration of 20ng/mL, and PDGF at a final concentration of 80 ng/mL.
The final concentration ratio of CHIR99021, VEGF, SCF, IL-3 and CSF in the second stage culture medium is 0.25: 1.0: 0.625, and the specific formula is as follows: to E6 basal medium, CHIR99021 was added at a final concentration of 2. mu.M, VEGF at a final concentration of 80ng/mL, Stem Cell Factor (SCF) at a final concentration of 80ng/mL, interleukin-3 (IL-3) at a final concentration of 50ng/mL, and CSF at a final concentration of 50 ng/mL.
The final concentration ratio of VEGF, SCF, HGH, Flt3L, EPO, EGF, UM171/UM729 in the third stage medium was 1.0: 1.25: 0.5: 1.0: 2.0: 0.2, the specific formula is as follows: VEGF at a final concentration of 40ng/mL, Stem Cell Factor (SCF) at a final concentration of 50ng/mL, HGH at a final concentration of 20ng/mL, Flt3 at a final concentration of 20ng/mL, EPO at a final concentration of 40ng/mL, EGF at a final concentration of 40ng/mL, and UM171/UM729 at a final concentration of 35. mu.M were added to Stemline II basal medium.
The method for inducing differentiation of embryonic stem cells (ES) into hematopoietic stem cells by the cytokine method used in this example includes:
(1) treating the Aggrewell 24-well plate with AARS to make the Aggrewell plate suitable for seeding embryonic stem cells and ensure that no adherence occurs and embryoid bodies are formed;
(2) the inoculation culture medium in the step (1) uses a first-stage differentiation culture medium, and the inoculation quantity is 30 ten thousand cells per hole;
(3) the day of inoculation was designated as day 0, and the embryos were cultured in a 5% CO2 incubator at 37 ℃ for 4 days, and at day 4, mature and well-conditioned embryoid bodies were selected under a stereomicroscope, and inoculated into T75 flasks per 150 embryoid bodies, and cultured in a 5% CO2 incubator at 37 ℃ for 4 days using a second-stage differentiation medium. And (3) beginning to change the culture medium on the 6 th day, removing the second-stage differentiation culture medium by suction, adding a third-stage differentiation culture medium, changing the culture medium by half every 3 days to improve the differentiation efficiency, wherein the third-stage differentiation period is 7-20 days, and collecting hematopoietic stem cells on the 12 th day in the embodiment.
For comparison, this example also provided a control group, which differs from this example only in that the first induced differentiation was carried out using only the following formulation of the medium: to the E8 complete medium was added Stem Cell Factor (SCF) at a final concentration of 40ng/mL, recombinant human granulocyte colony-stimulating factor (rhG-CSF) at a final concentration of 40ng/mL, VEGF at a final concentration of 10ng/mL, and PDGF at a final concentration of 40 ng/mL.
Example 2
This example is a modification of example 1, and is modified only in that induced pluripotent stem cells (iPS) are selected as the type of pluripotent stem cells.
For comparison, this example also provided a control group, which differs from this example only in that the first induced differentiation was carried out using only the following formulation of the medium: to the E8 complete medium was added Stem Cell Factor (SCF) at a final concentration of 40ng/mL, recombinant human granulocyte colony-stimulating factor (rhG-CSF) at a final concentration of 40ng/mL, VEGF at a final concentration of 10ng/mL, and PDGF at a final concentration of 40 ng/mL.
Results of examples 1-2
Detection of CD34+ positivity: the cells finally obtained in example 1, example 2 and the comparative group were collected, centrifuged at 1000r/min for 5min in a 50mL centrifuge tube, the supernatant was discarded, resuspended in 1mL PBS containing 2% (v/v) serum, the antibody CD34-PE-Cy7 was added, incubated at 4 ℃ for 30min, and washed once with PBS containing 2% serum. Then at 5X 106The cells were resuspended at a density of/ml and tested and analyzed for differentiation efficiency using an Agilent NovoCyte flow cytometer. In the detection process, the normal ES cells were used as a negative group, i.e., cells that were not subjected to hematopoietic differentiation induction or cells that were subjected to differentiation experiments but were not stained with flow antibody.
Results example 1 and comparative test results are shown in fig. 1, 4 and 5, and flow cytometry analysis found that the CD34+ positive rate of example 1 added with UM171 increased to 88.54% (for comparative example 60.21%), and that the fold of differentiation expansion of example 1 quantitatively added with UM171 was 98.4 fold (for comparative example 62.2 fold); the CD34+ positive rate of example 1 with UM729 added increased to 71.88% (60.61% in the control group), and the fold of differentiation expansion of example 1 quantitatively with UM729 added was 84.4 fold (44.4 fold in the control group); the results show that the differentiation efficiency of example 1 was significantly increased compared to its control group. The test results of example 2 and its comparative group are shown in fig. 2, fig. 4 and fig. 5, the CD34+ positive rate of example 2 added with UM171 is increased to 70.37% (its comparative group is 44.61%), and the differentiation expansion fold of example 2 quantitatively added with UM171 is 89.6 times (its comparative group is 59.4 times); the CD34+ positive rate of example 2 with UM729 added increased to 82.23% (39.20% in the control), and the fold expansion of differentiation of example 2 quantitatively with UM729 added was 75.8 fold (40.4 fold in the control); the results show a significant increase in differentiation efficiency of example 2 compared to its control group. Negative control group is shown in FIG. 3
The technical features of the embodiments described above may be arbitrarily combined, and for the sake of brevity, all possible combinations of the technical features in the embodiments described above are not described, but should be considered as being within the scope of the present specification as long as there is no contradiction between the combinations of the technical features.
The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.

Claims (10)

1. A medium for increasing the efficiency of differentiation of pluripotent stem cells into hematopoietic stem cells, comprising: comprises a first stage culture medium, a second stage culture medium and a third stage culture medium;
the first stage culture medium comprises an E8 complete culture medium, SCF, rhG-CSF, VEGF and PDGF, and the final concentration ratio of the SCF, the rhG-CSF, the VEGF and the PDGF in the first stage culture medium is (1.0-2.0) to (0.25-0.5) to (1.0-2.0);
the second stage culture medium comprises an E6 basal culture medium, CHIR99021, VEGF, SCF, IL-3 and CSF, and the final concentration ratio of the CHIR99021, the VEGF, the SCF, the IL-3 and the CSF in the second stage culture medium is (0.25-1.0): (1.0-2.0): (0.5-2.0);
the third stage culture medium comprises a Stemline II basal culture medium, SCF, HGH, Flt3L, EPO, EGF and novel pyrimidine indole compounds, and the final concentration ratio of the SCF, the HGH, the Flt3L, the EPO, the EGF and the novel pyrimidine indole compounds in the third stage culture medium is (0.5-2.0): (0.2-0.4): (0.5-2.0): (1.0-2.0): (0.2-0.4).
2. The medium of claim 1, wherein the novel pyrimidine indole compound comprises one or both of UM171 and UM 729.
3. The medium according to claim 1, wherein the final concentration of SCF, rhG-CSF, VEGF, and PDGF in the first-stage medium is 50-100ng/mL, rhG-CSF is 50-100ng/mL, VEGF is 10-50mM/mL, and PDGF is 50-100 ng/mL; the final concentration of CHIR99021 in the second stage culture medium is 1-5 mu M, VEGF and is 50-100ng/mL, the final concentration of SCF is 20-80ng/mL, the final concentration of IL-3 is 20-80ng/mL, and the final concentration of CSF is 20-80 ng/mL; the final concentration of SCF in the third stage culture medium is 20-80ng/mL, the final concentration of HGH is 10-100ng/mL, the final concentration of Flt3L is 20-80ng/mL, the final concentration of EPO is 20-80ng/mL, the final concentration of EGF is 20-80ng/mL, and the final concentration of the novel pyrimidine indole compound is 10-50 mu M.
4. The medium of claim 1, wherein the final concentration ratio of SCF, rhG-CSF, VEGF, PDGF in the first stage medium is 1.0: 0.25: 1.0; the final concentration ratio of CHIR99021, VEGF, SCF, IL-3 and CSF in the second-stage culture medium is 0.25: 1.0: 0.625; the final concentration ratio of SCF, HGH, Flt3L, EPO, EGF and the novel pyrimidine indole compound in the third stage culture medium is 1.0: 0.5: 1.0: 2.0: 0.2.
5. The medium of claim 1, wherein the final concentration of SCF, rhG-CSF, VEGF, and PDGF in the first phase medium is 80ng/mL, rhG-CSF is 80ng/mL, VEGF is 20ng/mL, and PDGF is 80 ng/mL; the final concentration of CHIR99021 in the second-stage culture medium is 80ng/mL for a final concentration of 2 mu M, VEGF, 80ng/mL for a final concentration of SCF, 50ng/mL for a final concentration of IL-3 and 50ng/mL for a final concentration of CSF; the final concentration of VEGF in the third stage culture medium is 40ng/mL, the final concentration of SCF is 50ng/mL, the final concentration of HGH is 10ng/mL, the final concentration of Flt3L is 20ng/mL, the final concentration of EPO is 40ng/mL, the final concentration of EGF is 40ng/mL, and the final concentration of the novel pyrimidine indole compound is 35 mu M.
6. A method of increasing the efficiency of differentiation of pluripotent stem cells into hematopoietic stem cells comprising the steps of:
s1: placing pluripotent stem cells in an Aggrewell plate, and performing first-stage induced differentiation into embryoid bodies in the first-stage medium according to any one of claims 1 to 5;
s2: placing the embryoid body in a second-stage culture medium of any one of claims 1 to 5 for a second-stage induced differentiation;
s3: placing the cell mixture after induced differentiation of S2 in the third-stage medium according to any one of claims 1 to 5, and performing third-stage induced differentiation.
7. The method according to claim 6, wherein the first stage culture of inoculating pluripotent stem cells on an Aggrewell plate for embryoid body differentiation is designated as day 0, the induced differentiation of the first stage starts from day 0 to day 4, the induced differentiation of the second stage starts from day 4 to day 6, and the induced differentiation of the third stage starts from day 8 to day 12.
8. The method according to claim 6, wherein the first stage differentiation-inducing culture medium is used for the inoculation period from day 0 to day 4, during which the solution is not changed; transferring the embryoid bodies to a T75 culture bottle for culture to the 6 th day on the 4 th day of the second stage of induced differentiation, and adding a second stage of differentiation culture medium during the transfer period without changing the medium; on the 6 th day of the third stage of induced differentiation, the medium in the T75 flask was replaced with fresh medium for the third stage, and half every 3 days from the 6 th day of the third stage of induced differentiation was replaced with fresh medium for the third stage.
9. The method for improving the efficiency of differentiation of pluripotent stem cells into hematopoietic stem cells according to claim 6, wherein the number of pluripotent stem cells seeded in the Aggrewell plate is 30 ten thousand cells per well.
10. The method of claim 6, wherein the pluripotent stem cells comprise one or both of induced pluripotent stem cells and embryonic stem cells.
CN202110652831.7A 2021-06-11 2021-06-11 Culture medium and method for improving efficiency of differentiation of pluripotent stem cells into hematopoietic stem cells Pending CN113373114A (en)

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