CN113249320B

CN113249320B - Application of abscisic acid in promoting in-vitro differentiation of human induced pluripotent stem cells into megakaryocytes and platelets

Info

Publication number: CN113249320B
Application number: CN202110576129.7A
Authority: CN
Inventors: 顾海慧; 钱宝华; 李彦欣; 黄韦华
Original assignee: First Affiliated Hospital of Naval Military Medical University of PLA
Current assignee: First Affiliated Hospital of Naval Military Medical University of PLA
Priority date: 2021-05-26
Filing date: 2021-05-26
Publication date: 2022-12-16
Anticipated expiration: 2041-05-26
Also published as: CN113249320A

Abstract

The invention relates to the technical field of medicines, and provides application of abscisic acid in preparing a promoter for in vitro differentiation of human induced pluripotent stem cells into megakaryocytes and platelets, wherein the optimal concentration of the abscisic acid in the promoter is 10 mu mol/L. In addition, the invention also provides a method for improving the in-vitro megakaryocyte differentiation efficiency of the human induced pluripotent stem cells, in the process of inducing and differentiating the megakaryocytes by the human induced pluripotent stem cells, abscisic acid is added at least at 0-14 days for stimulation, and through experimental verification, ABA promotes the generation and maturation of human iPSC-derived MK; ABA stimulation of human iPSCs in differentiation culture at 0-14 days can promote the generation quantity of megakaryocytes to be increased, and ABA stimulation is only carried out at 14-19 days, so that the increase and differentiation of MK quantity are not promoted; on day 19, the effect of continuously stimulating 0-14 days of ABA in differentiation culture of human iPSCs on MK differentiation is equivalent to the effect of continuously stimulating 0-19 days of human iPSCs in differentiation culture.

Description

Application of abscisic acid in promoting in-vitro differentiation of human induced pluripotent stem cells into megakaryocytes and platelets

Technical Field

The invention belongs to the field of biological medicine, and particularly relates to application of abscisic acid in promoting in-vitro differentiation of human induced pluripotent stem cells into megakaryocytes and platelets, and a method for improving in-vitro megakaryocyte differentiation efficiency of the human induced pluripotent stem cells.

Background

Platelet transfusion is an important supportive treatment for patients after major operations and radiotherapy and chemotherapy, and is an irreplaceable treatment method for bleeding caused by thrombocytopenia or dysfunction. At present, platelets used for clinical treatment can only be obtained through volunteer compensation donation, the blood source is extremely tight, and the risk of blood transfusion reaction and infection of diseases transmitted by menstrual blood exists. Therefore, the clinical supply of platelets is always in a state of shortage, especially for patients with difficult typing and production of Human Leukocyte Antigen (HLA) or Human Platelet Antigen (HPA) antibodies.

Induced Pluripotent Stem Cells (iPSCs) are stem cells cultured by reprogramming somatic cells, and can be infinitely expanded and cultured in vitro; human iPSCs are expanded in vitro, induced and differentiated into megakaryocytes and generate platelets, so that the shortage of clinical platelet supply can be relieved, and the platelet supply of patients with matching difficulty can be guaranteed.

Although many laboratories have successfully reported that the induced pluripotent stem cells are used for inducing differentiation of megakaryocytes in vitro and producing platelets, the efficiency of platelets produced in vitro from human iPSCs-derived megakaryocytes is less than 1/1000 of that produced in vivo, the functions of the produced platelets in vivo are reduced, and the price of the platelets is far higher than that of donated platelets. Therefore, methods for inducing pluripotent stem cells to differentiate megakaryocytes in vitro still need to be further improved.

Abscisic acid (ABA) is an important plant hormone that protects plants from biotic and abiotic stress stimuli. Although plants and humans have completely different ABA receptors, several studies have shown that ABA also has a universal signaling molecule in plants and animals. It has been shown that human granulocytes, mesenchymal Stem Cells (MSCs) and beta pancreatic cells can produce and release ABA and exert cell-specific effects. In an in vivo model, ABA was shown to increase intracellular Ca2 ⁺ The concentration expands the number of human non-committed hematopoietic progenitor cells (HPs). However, many factors for the production of Megakaryocytes (MKs) and platelets from stem cells are not currently determined. It is not clear whether stimulation of ABA action from early embryonic stage is beneficial for in vitro iPSC-derived MK differentiation/maturation. The effect of ABA on Hematopoietic Progenitor Cells (HPCs) and MK production by human ipscs is worthy of investigation.

Disclosure of Invention

The invention is based on the research, and explores the promotion effect of abscisic acid on the in vitro differentiation of human induced pluripotent stem cells into megakaryocytes and platelets, the first purpose is the application of abscisic acid in preparing a promoter for the in vitro differentiation of human induced pluripotent stem cells into megakaryocytes and platelets, and the second purpose is to provide a method for improving the in vitro megakaryocyte differentiation efficiency of human induced pluripotent stem cells, and improve the megakaryocyte differentiation efficiency and the number of platelets.

Normal human platelets are directionally differentiated into multipotent progenitors (MPP), myeloid progenitors (CMP) and megakaryoerythroid progenitors (MEP) from Hematopoietic Stem Progenitors (HSPCs), megakaryocytes (MK) are generated from megakaryocytes (Promegakarycyte), megakaryocytes mature to form primitive platelets (proplatiet), and finally fall off in blood vessels to form functional platelets.

The in vitro induction of induced pluripotent stem cells (iPS) to generate platelets is regulated and controlled in two stages: (1) iPS cells are differentiated into hematopoietic stem and progenitor cells; (2) hematopoietic stem progenitor cells are directionally differentiated into megakaryocytes and platelets.

Abscisic acid (ABA) is a naturally occurring phytohormone, a safe and non-toxic substance. Abscisic acid stimulates the activity of innate immune cells, mesenchymal stem cells and hematopoietic stem cells, as well as insulin-releasing pancreatic beta cells in mammals through the second messenger cyclic adenosine monophosphate signaling pathway. Micromolar amounts of abscisic acid have been shown to proliferate undifferentiated human hematopoietic progenitor cells.

The invention proves the function of abscisic acid in inducing the differentiation of the megakaryocytes of the pluripotent stem cells in vitro for the first time, and the abscisic acid is a proliferation factor for inducing the differentiation of the megakaryocytes of the pluripotent stem cells in vitro.

The application of abscisic acid can increase the in vitro differentiation of human induced pluripotent stem cells into CD34 ⁺ /CD45 ⁺ HPCs, promote the production of Megakaryocytes (MKs) and platelets. ABA PKA-dependent activation of CD34 by the receptors LANCL2 and GRP78 ⁺ /CD45 ⁺ The Akt and ERK1/2 signaling pathways on HPCs function.

The inventor finds that ABA treatment promotes hematopoietic progenitor cells (CD 34) at day 14 in-vitro differentiation culture of human iPSCs ⁺ /CD45 ⁺ HPCs) and megakaryocytes (CD 41) ⁺ MKs) production, with increased CD41 by day 19 ⁺ /CD42b ⁺ Production of MKs and platelets.

In platelet derivation, abscisic acid treatment not only promoted CD41 at day 14 ⁺ Production of MKs and increased CD41 by day 19 ⁺ /CD42a ⁺ And CD41 ⁺ /CD42b ⁺ MKs and platelets. Although addition of abscisic acid on days 14-19 may promote production of MKs, addition of abscisic acid on days 0-14 may still be necessary.

In a first aspect, the invention provides the use of abscisic acid in the preparation of a promoter for the in vitro differentiation of human induced pluripotent stem cells into megakaryocytes and platelets.

Preferably, the concentration of abscisic acid in the accelerant is 10-25 mu mol/L, and the optimal concentration is10. Mu. Mol/L. Different concentrations of ABA were added to differentiation medium and CD34 was detected by flow cytometry at day 14 ⁺ /CD45 ⁺ HPCs and CD41 ⁺ Generation of MKs; the results show that CD41 is present at an ABA concentration of 10. Mu.M ⁺ The highest number of MKs was determined, and 10 μ MABA was probably the optimal concentration to stimulate megakaryocyte differentiation (fig. 1).

In a second aspect of the present invention, there is provided a method for increasing the efficiency of differentiation of megakaryocytes in vitro from human induced pluripotent stem cells: in the process of inducing differentiation of megakaryocytes by human induced pluripotent stem cells, abscisic acid is added at least on days 0 to 14 for stimulation.

Preferably, the concentration of the abscisic acid is 10 mu mol/L. In the induced differentiation process, abscisic acid is added for stimulation only on days 0-14.

Through experimental verification, the ABA can promote the MK differentiation stage to advance, but only at the 14 th to 19 th days, the ABA stimulation can not promote the MK differentiation; stimulating and promoting iPSCs to differentiate into MK on days 0-14 of ABA, wherein the promoting effect is equivalent to the stimulating effect of days 0-19; meanwhile, ABA stimulation can promote megakaryocyte differentiation and maturation, but the action effect is better than that of ABA continuous stimulation in 0-19 days.

Further, the method for improving the differentiation efficiency of the human induced pluripotent stem cells in vitro megakaryocytes comprises the following specific steps:

(1) Day-3: inducing human pluripotent stem cells to 5-10X 10 ⁴ The density of/mL is inoculated in a six-hole plate pretreated by Matrigel, and a Saeby culture medium is used for culture;

(2) Day 0: digesting the cells into single cells after the cell overgrowth is more than 70 percent; adding the solution to a U-bottom 96-well plate at 3000-4000 cells/well with 50. Mu.L serum-free medium per well, centrifuging at 1500rpm,5min, standing at 37 deg.C, and 5% CO ₂ Culturing in an incubator; the serum-free culture medium contains 10ng/mL BMP4, 10ng/mLbFGF, 10 mu M ROCK inhibitor Y27632 and 10 mu mol/L abscisic acid;

(3) Day 2: supplementing 50 μ L/Kong Jiaye with serum-free medium containing 10ng/mL BMP4, 10ng/mL FGF, 20ng/mL EGF, 50ng/mL SCF and 10 μmol/L abscisic acid;

(4) Day 5: supplementing 50 μ L/Kong Jiaye with serum-free medium containing 10ng/mL BMP4, 10ng/mL FGF, 10ng/mL EGF, 50ng/mL SCF and 10 μmol/L abscisic acid;

(5) Day 8: discard 100. Mu.L of liquid per well and add 50. Mu.L of serum-free medium containing 10ng/mLBMP4, 10ng/mLbFGF, 10ng/mLVEGF, 50ng/mL SCF and 10. Mu. Mol/L abscisic acid per well;

(6) Day 11: the broth was supplemented with 50. Mu.L of serum-free medium/well containing 10ng/mLBMP4, 10ng/mLbFGF, 10ng/mLVEGF, 50ng/mL SCF, 60ng/mL TPO and 10. Mu. Mol/L abscisic acid at a final TPO concentration of 20ng/mL;

(7) Day 14: collecting hematopoietic cells secreted by EB cells, transferring the cells to a 6-hole plate for culture, and continuously culturing for 5 days, wherein the culture solution is 2mL of serum-free culture medium/hole; the serum-free culture medium contains 20ng/mL SCF, 10ng/mL IL 11, 50ng/mL TPO and 10 mu mol/L abscisic acid;

(8) Day 19: megakaryocytes were collected.

Further, the method for improving the in vitro megakaryocyte differentiation efficiency of the human induced pluripotent stem cells further comprises the steps of reviving the human induced pluripotent stem cells and subculturing,

the cell recovery process comprises the following steps: (1) preheating a cell culture solution in a 37 ℃ water bath; (2) Taking out the cell freezing tube from liquid nitrogen or a refrigerator at minus 80 ℃, immediately shaking the cell freezing tube in a water bath kettle at 37 ℃ to quickly unfreeze cells until Yu Xiao ice cakes remain in the liquid, quickly taking out the cell freezing tube, transferring the cell freezing tube to a super clean bench, adding 1mL of culture solution, uniformly mixing, and centrifuging at 1500rpm for 5min; (3) Removing supernatant, adding preheated culture solution into the freezing tube, re-suspending cells, counting, and seeding into a 6-hole plate, and uniformly mixing the cells by a cross method; (4) Placing 6 well plates at 37 ℃ 5% CO ₂ And (5) incubation in an incubator.

The subculture process comprises the following steps: (1) preparing a matrigel-paved six-hole plate; (2) preheating the seashell culture solution in a water bath kettle at 37 ℃; (3) Taking the cell culture plate out of the incubator, observing the growth state and density of the cell culture plate under a microscope, and allowing the cell clone density to reach 70-80% for passage; (4) Placing a culture dish of the human induced pluripotent stem cells in a super clean bench, removing a supernatant by using an aseptic pipette, taking 1ml PBS, washing the cells once in the culture dish, and removing PBS; (5) Adding 1mL0.05mM EDTA into a culture dish, slightly shaking to fully pave the EDTA on the surface of cells, observing the edge of cell clone under a mirror to roll up, enabling the cells to become round, absorbing the EDTA by using a pipette gun, adding 2ml of Saebi culture solution by using the pipette gun, slightly blowing down the cells at the bottom of the culture dish, and carrying out passage to a 6-well plate paved with Matrigel at different densities according to the cell clone density; (6) And observing the cell density after 24h, and carrying out next passage when the cell clone density reaches 70-80%.

Action and Effect of the invention

Through experimental verification, ABA promotes generation and maturation of human iPSC-derived MK; ABA stimulation of human iPSCs in differentiation culture at 0-14 days can promote differentiation stage of megakaryocytes to be advanced, and only in 14-19 days, ABA stimulation is carried out, and MK differentiation is not promoted; on day 19, the effect of continuously stimulating 0-14 days of ABA in differentiation culture of human iPSCs on MK differentiation is equivalent to the effect of continuously stimulating 0-19 days of human iPSCs in differentiation culture.

Mechanistically, there was no significant difference in ERK1/2 and AKT signaling at day 8 in the presence of ABA, but phosphorylation of AKT and ERK1/2 involved in MK differentiation was significantly increased at 14 days relative to the ABA-untreated group, indicating that the level of activation of ERK1/2 and AKT signaling pathways was enhanced relative to the untreated group with 14 days of ABA stimulation.

Therefore, abscisic acid has great potential in promoting the in vitro differentiation of human induced pluripotent stem cells into megakaryocytes and platelets, and the invention also provides a new way for obtaining platelets in vitro.

Drawings

FIG. 1 shows the effect of different abscisic acid concentrations on the in vitro differentiation of iPSCs, A. CD produced at different culture time periods ³⁴ ⁺ /CD ^45- HSCs、CD ³⁴⁺ /CD ⁴⁵⁺ HPCs、CD ⁴¹⁺ Percentage of MKs; B. on day 14 of differentiation, different concentrations of ABA (0. Mu.M, 1. Mu.M, 2.5. Mu.M, M,5. Mu.M, 10. Mu.M, 15. Mu.M, 20. Mu.M, 25. Mu.M) stimulation of CD ³⁴⁺ /CD ⁴⁵⁺ HPCs and CD ⁴¹⁺ Relative count ratio of MKs. (. P)<0.01；*P<0.05)。

FIG. 2 shows the cell number measurements at day 19 of differentiation, A is the total number of cells compared between ABA-treated and non-treated groups (P < 0.05); and B is the comparison of ABA treated group and non-treated group under an ordinary microscope at the 19 th day.

FIG. 3 shows MK flow assay results at day 19: A. day 19, ABA treatment group flow analysis representative; relative CD at day 19 in both B-D.ABA untreated and treated groups ⁴¹⁺ ，CD ⁴¹⁺ CD ^42a+ And CD ⁴¹⁺ CD ^42b+ Cell count (. About.P)<0.001；**P<0.01)。

FIG. 4 shows the megakaryocyte morphology and DNA ploidy measurements: a.wright-Giemsa stained representative image. BM derived MK (left); hiPSC source MK (right); scale bar =25 μ ι η; B. on day 19, the expression of the hiPSC-MKs surface molecules vWF, CD42b and β -tubulin were examined by confocal laser microscopy. Cells were stained with antibodies against VWF (red), against CD42b (green), against DAPI (blue) (scale bar =25 μm); C. representative data for hiPSC-MKs DNA ploidy in ABA treated and untreated groups (P < 0.01;. P < 0.05) on day 19.

Fig. 5 is a platelet morphology observation: A. a typical picture of pre-platelets under normal light microscopy (scale bar =50 μm); B. on day 19, the number of MK-PLTs produced by the ABA non-treated group was compared to the treated group (counts under light microscope;. P < 0.01).

Fig. 6 is a comparison of cell counts at day 8: CD in ABA treated and untreated groups ³⁴⁺ /CD ⁴⁵⁺ ，CD ³⁴⁺ /CD ^45- Relative cell count (. About.P)<0.001)。

FIG. 7 is a cell number measurement at day 14: A. total cell numbers released from hiPSC-EBs (P) in ABA-treated and non-treated groups at day 14<0.01 ); B-D.on day 14, relative CD in ABA-treated and untreated groups ³⁴⁺ CD ⁴⁵⁺ ，CD ³⁴⁺ Cd ^45- And CD ⁴¹⁺ Cell count (. About.P)<0.001；**P<0.01；*P<0.05 ); E. analysis by flow cytometryGating strategies for cultured HPC and MK.

FIG. 8 is day 14, cell staining observations: wright-Giemsa stained representative HPC images (scale bar =10 μm); B. on day 14, the expression of the hiPSC-HPCs surface molecules CD34, CD456 were examined by ABA-treated and non-treated laser confocal microscopy. Cells were stained with antibodies against CD34 (green), CD45 (red), DAPI (blue) (scale bar =25 μm).

FIG. 9 is a collection of cells at day 14 for colony formation experiments: A. on day 14, CFC counts from ABA-treated and non-treated groups were compared (P <0.01; P < 0.05); B. representative images of different CFUs (scale bar =25 μm).

FIG. 10 is a comparison of total cell number (P > 0.05) at day 19 between non-ABA-treated and ABA (d 14-19) -treated groups.

FIG. 11 is MK detection at day 19: CD in A-C, untreated and ABA (d 14-19) treated groups ⁴¹⁺ ，CD ⁴¹⁺ /CD ^42a+ And CD ⁴¹⁺ /CD ^42b+ Relative cell counts; D. day 19, the non-treated group was compared with the ABA (d 14-19) treated group for the amount of MK-PLTs produced (counting under the light microscope).

FIG. 12 shows the total cell count of the non-ABA-treated group compared to the ABA (d 0-14) and ABA (d 0-19) treated groups at day 19 (. About.P < 0.01;. About.P < 0.05).

FIG. 13 is day 19, MK flow analysis: day 19, untreated, ABA (d 0-14) and ABA (d 0-19) treated groups CD ⁴¹⁺ ，CD ⁴¹⁺ /CD ^42a+ And CD ⁴¹⁺ /CD ^42b+ Relative counting of cells; D. day 19, comparison of MK-PLTs (counted under a light microscope) was generated in the non-treated group, ABA (d 0-14) treated group and ABA (d 0-19) treated group; E. comparison of MK production per HPC in untreated, ABA (d 0-14), ABA (d 14-19) and ABA (d 0-19) treated groups (. SP. < 0.001;. SP. < 0.01;. SP. < 0.05).

Figure 14 western blot analysis and flow cytometry analysis: a.s production Process CD was determined by Western blotting ³⁴⁺ /CD ^45- HSCs、CD ³⁴⁺ /CD ⁴⁵⁺ HPCs and CD ⁴¹⁺ Proteins of LANCL2 and GRP78 in MKsExpression level (. About.P)<0.001**P<0.01 ); B. immunoblotting for determination of CD in untreated group and ABA-treated group ³⁴⁺ /CD ^45- HSCs、CD ³⁴⁺ /CD ⁴⁵⁺ HPCs and CD ⁴¹⁺ Expression levels of AKT, P-AKT, ERK1/2 and P-ERK1/2 in MKs (. About.P)<0.001,**P<0.01 ); C. total cell counts (. Times.P) at day 19 after different concentrations of H89 treatment (0. Mu.M, 2.5. Mu.M, 5. Mu.M and 10. Mu.M) in untreated or ABA-treated groups<0.001)；**P<0.01). (D-E) CD after different concentrations of H89 treatment (0. Mu.M, 2.5. Mu.M, 5. Mu.M and 10. Mu.M) in the untreated group or ABA-treated group on day 19 ⁴¹⁺ Cells and CD ⁴¹⁺ /CD ^42b+ Percentage of cells (. About.P)<0.01；*P<0.05 ); F. detection of CD (0. Mu.M, 2.5. Mu.M, 5. Mu.M and 10. Mu.M) in untreated or ABA-treated groups after treatment with different concentrations of H89 by Western blotting ³⁴⁺ /CD ⁴⁵⁺ Expression levels of AKT, P-AKT, ERK and P-ERK in HPCs (. About.P)<0.001)。

FIG. 15 shows the effect of ABA on the phosphorylation of AKT and ERK1/2 involved in MK differentiation by stimulation on days 0-14.

Detailed Description

The present invention will be described in detail below with reference to examples and the accompanying drawings. The following examples should not be construed as limiting the scope of the invention.

1. Experimental methods

(I) cell recovery

1. Preparing an ice box and a 37 ℃ water bath kettle;

2. preheating the cell culture solution in a 37 ℃ water bath;

3. removing the cell freezing tube from liquid nitrogen or a refrigerator at-80 deg.C, and placing on ice;

4. taking the freezing tube, immediately shaking the freezing tube in a water bath kettle at 37 ℃ to quickly unfreeze cells, quickly taking the cells out when Yu Xiao ice cubes exist in the liquid, transferring the cells to a super clean bench, adding 1mL of culture solution, uniformly mixing, and centrifuging at 1500rpm for 5min;

5. removing supernatant, adding preheated culture solution into the freezing tube, re-suspending cells, counting, and seeding into a 6-hole plate, and uniformly mixing the cells by a cross method;

6. placing 6 well plates at 37 ℃ 5% CO ₂ IncubatorAnd (4) incubating.

(II) culture and passage of CBiPS cells

1. Placing a pipette, a 15mL centrifuge tube and a 6-hole cell culture plate in a super clean bench, and performing ultraviolet disinfection for 30min;

2. preparing a 37 ℃ water bath kettle;

3. preparing a six-hole plate for bedding matrigel;

4. preheating the scallop culture solution in a water bath kettle at 37 ℃;

5. taking the CBiPS cell culture plate out of the incubator, observing the growth state and density of the CBiPS cell culture plate under a microscope, wherein the cell clone density reaches 70-80% and can be passed;

6. placing the CBiPS cell culture dish in a super clean bench, removing a supernatant by using a sterile pipette, taking 1mLPBS, washing cells in the culture dish once, and removing PBS;

7. adding 1mL of 0.05mM EDTA into a culture dish, slightly shaking to fully pave the EDTA on the surface of cells, observing the edge of the CBiPS cell clone under a mirror to roll up, enabling the cells to become round, absorbing the EDTA by using a pipette, adding 2ml of seebeck culture solution by using the pipette, slightly blowing down the cells at the bottom of the culture dish, and carrying out passage to a 6-well plate paved with Matrigel at different densities according to the cell clone density;

and observing the cell density after 8.24h, and carrying out next passage when the cell cloning density reaches 70-80%.

(III) culturing induced differentiation megakaryocyte by hiPSCs Spin-EB method

1. Day-3: according to 5-10X 10 ⁴ density/mL iPS cell species were cultured in Matrigel pretreated six-well plates using seebeck medium.

2. Day 0: after the cells were overgrown with > 70%, iPS cells were digested into single cells with Accutase, the solution was added to a U-bottom 96-well plate at 3000-4000 cells/well and 50. Mu.L/well Serum Free Media (SFM), followed by centrifugation at 1500rpm for 5min, and cultured at 37 ℃ in a 5-vol CO2 incubator; SFM medium was supplemented with BMP4 (10 ng/ml), bFGF (10 ng/ml) and ROCK inhibitor (Y27632, 10. Mu.M).

3. Day 2: the aliquots were supplemented with 50 μ LSFM/well, SFM containing BMP4 (10 ng/ml), bFGF (10 ng/ml), VEGF (20 ng/ml) and SCF (100 ng/ml); final cytokine concentration: BMP4 (10 ng/ml), bFGF (10 ng/ml), VEGF (10 ng/ml) and SCF (50 ng/ml).

4. Day 5: make up 50 μ LSFM/well, SFM containing BMP4 (10 ng/ml), bFGF (10 ng/ml), VEGF (10 ng/ml) and SCF (50 ng/ml);

5. day 8: discard 100. Mu.L of liquid per well, and add 50. Mu.L of SFM containing BMP4 (10 ng/ml), bFGF (10 ng/ml), VEGF (10 ng/ml) and SCF (50 ng/ml);

6. day 11: supplementing with 50 μ LSFM/well SFM containing BMP4 (10 ng/ml), bFGF (10 ng/ml), VEGF (10 ng/ml), SCF (50 ng/ml) and TPO (60 ng/ml), wherein the final concentration of TPO is 20ng/ml;

7. day 14: hematopoietic cells secreted by EB cells (CD 34) were collected ⁺ ) The cells were transferred to a 6-well plate and cultured in 2 mL/well of SFM containing SCF (20 ng/mL), IL 11 (10 ng/mL) and TPO (50 ng/mL), and the culture was continued for 5 days.

10 μ MABA was added to the cells at the same time for each fluid infusion, and megakaryocytes (CD 41) were collected on day 19 ⁺ )。

(IV) observation of megakaryocyte morphology

The hPSCs of the ABA treated group and the non-treated group were observed by an ordinary optical microscope to induce differentiated megakaryocytes, and the cell morphology and the number were compared.

(V) megakaryocyte polyploidy assay

Suspension cells cultured for 19 days were collected, washed by centrifugation, incubated with 25. Mu.g/ml Hoechst 33342 (Sigma-Aldrich) for 45 minutes at 37 ℃ and labeled with anti-human CD41a-APC and CD42b-FITC for 30 minutes, washed with pre-cooled PBS, and then subjected to flow analysis by adding PI.

(VI) flow assay

On

days

14 and 19, the harvested single cells were labeled anti-human CD34-PE (Invitrogen, www.thermofisher.com), CD45-APC-cy7 (Invitrogen), CD41-APC (eBioscience, http:// www (eBioscience. Com), CD42 a-effector 450 (eBioscience), CD42b-FITC (eBioscience.) all samples were analyzed by FACS Calibur flow cytometer (BD Biosciences).

(hepta) MK morphometric assay

After 19 days of differentiation, individual cells were stained with Wright-Giemsa for cell morphology analysis.

(VIII) confocal microscopy analysis

1. Collecting cells cultured for 19 days, and smearing the cells by using a cell smear machine;

2.4% paraformaldehyde, fixed for 20min (protected from light), followed by 3 washes with PBS (5 min/time);

TritonX-100 (0.3% -0.5%), permeabilized for 20min (protected from light), washed 3 times with PBS (5 min/time);

4.5% BSA blocking for 1h;

5. a first antibody: prepare 150 μ l of anti-vWF antibody (1;

6. pour out primary antibody, wash 3 times with PBS (5 min/time);

7. secondary antibody: 594 antibody (1; CD42b-FITC (1;

8, DAPI:1, 300 incubation for 5min, 3 times of PBS washing (5 min/time);

9. drying the glass slide, then dropwise adding a mounting agent, adding a cover slip to seal the glass slide, and slightly discharging air bubbles;

10. and (5) observing by using a confocal microscope.

2. Analysis of results

1. Application of ABA on days 0-19 to promote in vitro differentiation of human iPSCs, generation and maturation of MKs

1.1 selection of optimal concentration for ABA treatment

We performed hematopoietic differentiation based on the spin-EB differentiation system in feeder-free and serum-free conditions. CD34 released by EB on day 14 was collected ⁺ The cell population was cultured with further addition of rhTPO, IL-11 and SCF, and MK (CD 41) was obtained after 5 days ⁺ ) A cell.

To examine the effect of ABA, we first added different concentrations of ABA (0. Mu.M, 1. Mu.M, 2.5. Mu.M, 5. Mu.M, 10. Mu.M, 15. Mu.M, 20. Mu.M, 25. Mu.M) to the differentiation medium and then examined CD34 by flow cytometry at day 14 ⁺ /CD45 ⁺ HPCs and CD41 ⁺ Production of MKs. As shown in FIG. 1, although the response of HPCs to 10. Mu.M and 25. Mu.MABA was higher than that of ABA at other concentrations, CD was observed at 10. Mu.M ⁴¹⁺ The number of MKs is higher and therefore 10 μ MABA may be the optimal concentration to stimulate megakaryocyte differentiation.

1.2ABA treatment promoted proliferation of cells differentiated from hipscs

The cells collected on day 19 were counted and the results are shown in figure 2, with an increase in total cell number obtained on day 19 in the ABA treated group (figure 2a. When cells of day 19 ABA-treated and non-treated groups were observed simultaneously using an optical microscope, the number of cells was significantly greater in the ABA-treated group than in the non-treated group at the same magnification (fig. 2B).

1.3 day 19 ABA treatment promoted megakaryocyte differentiation and maturation

In addition, we performed flow cytometry on day 19 to determine the cell characteristics (fig. 3A). CD41 in the case of treatment with ABA according to flow cytometry analysis ⁺ ，CD41 ⁺ CD42a ⁺ And CD41 ⁺ CD42b ⁺ Cell counts were significantly increased (FIGS. 3B-D; P, respectively)<0.001，P<0.01 and P<0.001 It shows that ABA can promote differentiation and maturation of megakaryocytes.

1.4 morphological and chromosomal ploidy analyses confirm that ABA promotes megakaryocyte maturation

On day 19, megakaryocytes were analyzed for morphology and chromosomal ploidy: FIG. 4A shows a representative image of Wright-Giemsa staining, with bone marrow-derived megakaryocytes to the left and hipSC-derived megakaryocytes to the right (scale bar =25 μm); fig. 4B shows the immunofluorescence expression of vWF, CD42B in MK derived from hipscs at day 19. Cells were stained with antibodies against VWF (red), CD42b (green), DAPI (blue) (scale bar =25 μm).

On day 19, the hiPSC-MKs DNA ploidy was significantly higher in ABA treated group than in untreated group (fig. 4C), and the cell morphology also significantly biased towards mature megakaryocytes.

1.5 day 19 platelets were observed under a microscope

Both ABA-treated and non-treated groups were observed under the mirror for formation of pre-platelets and platelets (fig. 5A). On day 19, the culture supernatants were microscopically observed and counted for the formation of platelet precursors or blastocysts of MKs (MK-PLTs), with higher MK-PLT numbers in ABA treated groups than in untreated groups, with statistical differences (fig. 5 p-restricted to 0.01.

2. ABA stimulation at day 0-14 promotes CD41 ⁺ Differentiation of (MK) cells

To further investigate which stage ABA plays a role in the differentiation process of human iPSCs megakaryocytes. We collected immature hematopoietic cells released from EBs at day 14 of differentiation in human iPSCs differentiation culture for analysis. The results show that 0-14 days ABA stimulation can promote CD41 ⁺ Differentiation of MK cells.

2.1ABA stimulation promotes HSC production

First, flow cytometry was used to detect the expression of CD34 and CD45 in two groups of cells on day 8 of differentiation, and the results showed that ABA-treated CD34 group was found ⁺ /CD45 ⁺ The cell number was significantly higher than that of the untreated group, and the difference was statistically significant (FIG. 6;P)<0.001). CD34 between two groups ⁺ /CD45 ^- The cell number difference was not statistically significant (FIG. 6). This result indicates that ABA can promote HSC production.

2.2 day 14 flow cytometry analysis showed that ABA can promote the MK differentiation stage to be advanced

On day 14, EB-released cells were collected and counted to show that the total cell number was significantly higher in ABA-treated groups than in non-treated groups, with statistical differences (fig. 7A). The cell surface marker for HPC is CD34 ⁺ /CD45 ⁺ And the HSC cell surface marker is CD34 ⁺ /CD45 ^- ，CD41 ⁺ Is a surface marker for MK. Flow cytometric analysis detected these three cell surface molecules (CD 34, CD45 and CD 41) (fig. 7E, F). The results show that ABA-treated group CD34 ⁺ /CD45 ⁺ And CD34 ⁺ /CD45 ^- The number of cell populations was higher than in the control group, and the differences were statistically significant (FIG. 7B, C<0.001，P<0.05). Meanwhile, compared with the control group, the ABA-treated group CD41 ⁺ The number of cell populations was significantly increased, and the differences were statistically significant (figure 7d<0.01). The result shows that the MK differentiation tendency of the ABA treatment group is obviously higher than that of the control group, and the ABA can enter the MK differentiation stage earlier than that of the control group.

2.3 early hematopoietic cells were observed by Wright-Giemsa staining at day 14

On day 14, suspension cells were harvested for Wright-Giemsa staining and an image of early hematopoietic cells was visualized under the mirror, as shown in FIG. 8A. Expression of the cell surface molecules CD34 and CD45 associated with HPC in both ABA-treated and untreated groups was also observed by confocal laser immunofluorescence staining (fig. 8B).

2.4 colony formation assay

Cells generated for 14 days were collected for colony formation experiments to study the ability of ABA to promote colony formation. The results show that ABA-induced CFU-M/MK and CFU-GM colony formation is higher than that of the control group (P <0.01 and P < -0.05, respectively). The results indicate that 0-14d of ABA sustained stimulation promotes differentiation of HPC to MK. (FIG. 9A, B)

These results indicate that 0-14 day ABA stimulation can promote early differentiation of HPCs into MK cells.

3. ABA stimulation at day 14-19 alone did not promote MK differentiation

To further verify whether stimulation of ABA promotes MK maturation, we treated with 10 μ MABA only on days 14-19 of human iPSCs differentiation culture.

3.1 ABA stimulation in 14-19 days can not promote total cell proliferation in 19 days

We first compared the cell counts at day 19 in the ABA-treated and non-treated groups, and the results are shown in fig. 10 with no significant difference between the two groups (P = 0.3446).

3.2 ABA stimulation of megakaryocyte differentiation failure at day 14-19

Day 19 cells were harvested for CD41 ⁺ ，CD41 ⁺ CD42a ⁺ And CD41 ⁺ CD42b ⁺ FACS analysis of cells. The results show that ABA treatment group Total CD41 ⁺ ，CD41 ⁺ /CD42a ⁺ And CD41 ⁺ /CD42b ⁺ None of the cell counts were higher than the untreated group (FIGS. 11A-C); at day 19, MK-PLTs in the culture supernatants were observed and counted under a microscope, and the number of MK-PLTs in the ABA-treated group was not significantly different from that in the control group (FIG. 11D). These results indicate that 14-19 days ABA stimulation does not promote MK maturation.

4. D0-14 day stimulation of ABA also promotes MK differentiation, and on day 19, the promoting effect is equivalent to that of D0-19 stimulation

To date, we have not found which stage of ABA treatment is more capable of promoting MK proliferation. Our previous studies suggest that day 0 to 14 ABA stimulation may be more effective in promoting proliferation of MK cells at day 19. To demonstrate this, we added ABA only on days 0-14, stopped ABA addition on days 14 to 19, and performed statistical analysis at 19 days.

4.1 0-14 day ABA stimulation can promote total cell proliferation at 19 days

On day 19, we first counted the total cell number of ABA (0-14) treated and non-treated groups and compared. The results show that: the total cell number of the ABA-treated group was significantly higher than that of the ABA non-treated group (fig. 12 p-straw 0.01); meanwhile, the total number of cells in the ABA (d 0-14) treated group was not statistically different from that in the ABA (d 0-19) treated group at day 19 (FIG. 12).

4.2 0-14 days ABA stimulation can promote megakaryocyte differentiation and maturation

On day 19, flow analysis showed: ABA (d 0-14) -treated group CD41 compared to untreated group ⁺ 、CD41 ⁺ /CD42a ⁺ And CD41 ⁺ /CD42b ⁺ Cell counts were all significantly elevated (FIGS. 13A-C; P)<0.001，P<0.01 and P<0.001 And there was no significant difference in the above cell counts between the ABA (d 0-14) and ABA (d 0-19) treated groups (FIGS. 13A-C).

On day 19, MK-PLTs were observed and counted microscopically in the culture supernatants, with statistically significant differences in the amount of MK-PLTs produced by ABA (D0-14) treated groups compared to control groups (FIG. 13D P-Ap-0.01), and not significantly different from the amount of MK-PLTs produced by ABA (D0-19) treated groups (FIG. 13D).

The number of MK produced per HPC on day 19 in the untreated, ABA (d 0-14), ABA (d 14-19) and ABA (d 0-19) groups was further analyzed and compared, and the results showed that CD41 produced between the groups ⁺ /CD42b ⁺ There was no significant difference in MK/HPC amounts (FIG. 13E).

4.3 At 14 days, after ABA stimulation, the activation level of ERK1/2 and AKT signal transduction pathways is enhanced

To further investigate the mechanism of action of ABA, we assayed CD34 by Western blotting ⁺ /CD45 ^- HSCs、CD34 ⁺ /CD45 ⁺ HPCs and CD41 ⁺ Expression of the ABA receptor LANCL-2 and ABA-binding protein GRP78 in MKs (FIG. 14A). GRP78 and LANCL-2 expression increased during maturation of HPC and MK, in CD34 ⁺ /CD45 ⁺ Peaking in HPCs and subsequent CD41 ⁺ Expression of MKs was gradually decreased (fig. 14A). Subsequently, we further investigated the signaling mechanism of MK in differentiated cells. Under the action of ABA, the CD34 is found ⁺ /CD45 ^- The activation signals of ERK1/2 or AKT in HSCs are not significantly different from those of the control group, but CD34 ⁺ /CD45 ⁺ The levels of AKT and ERK1/2 phosphorylation were significantly increased in HPC (fig. 14B), indicating an increased level of activation of signaling pathways under ABA.

At day 0-14 of differentiation, stimulation with 0. Mu.M, 2.5. Mu.M, 5. Mu.M and 10. Mu.M PKA inhibitors (H89), with or without 10. Mu.MABA, results of the 19 th day study found total cell number and CD41 ⁺ /CD42b ⁺ The percentage of cells was inhibited by dose-dependent H89 treatment (figure 14C, E). Although different concentrations of H89 vs CD41 ⁺ Percentage of cells had no effect (FIG. 14D), but CD41 ⁺ The number of cells decreased significantly as the number of total cells decreased. The specific mechanism by which ABA promotes HPCs differentiation was further demonstrated by the reduction in the levels of ERK1/2 and AKT phosphorylation in HPCs following the action of PKA inhibitor H89 (FIG. 14F). These data indicate that addition of ABA on days 0-14 leads to activation of the ERK1/2 and AKT signaling pathways of the PKA cascade by increasing expression of its receptors, LANCL-2 and GRP78 proteins, in HPCs, facilitating subsequent differentiation and maturation of MK.

In conclusion, ABA promotes CD34 ⁺ /CD45 ⁺ Proliferation of HPCs and CD41 ⁺ MK differentiation, a schematic representation of which is shown in FIG. 15.

The foregoing shows and describes the general principles, essential features, and advantages of the invention. It will be understood by those skilled in the art that the present invention is not limited to the embodiments described above, which are given by way of illustration of the principles of the present invention, and that various changes and modifications may be made without departing from the spirit and scope of the invention as defined by the appended claims. The scope of the invention is defined by the appended claims and equivalents thereof.

Claims

1. Use of abscisic acid in preparing human induced pluripotent stem cell in vitro differentiation into megakaryocyte and platelet promoter is provided.

2. Use according to claim 1, characterized in that:

wherein, in the accelerant, the concentration of abscisic acid is 10-25 mu mol/L.

3. Use according to claim 1, characterized in that:

wherein, in the accelerant, the concentration of the abscisic acid is 10 mu mol/L.

4. A method for improving the differentiation efficiency of human induced pluripotent stem cells in vitro megakaryocytes is characterized by comprising the following steps:

(2) Day 0: when the cell clone density is more than 70%, digesting the cells into single cells; according to 3000-4000 cells

Adding 50 μ L of serum-free culture medium into each well, adding the solution into U-shaped bottom 96-well plate, centrifuging at 1500rpm for 5min, placing at 37 deg.C and 5% CO ₂ Culturing in an incubator; the serum-free culture medium contains 10ng/mL BMP4, 10ng/mL bFGF, 10 mu M ROCK inhibitor Y27632 and 10 mu mol/L abscisic acid;

(3) Day 2: serum-free medium was supplemented at 50. Mu.L/Kong Jiaye, containing 10ng/mL

BMP4, 10ng/mL bFGF, 20ng/mLVEGF, 50ng/mL SCF and 10 μmol/L abscisic acid;

(4) Day 5: serum-free medium was supplemented at 50. Mu.L/Kong Jiaye, containing 10ng/mL

BMP4, 10ng/mL bFGF, 10ng/mLVEGF, 50ng/mL SCF and 10 μmol/L abscisic acid;

(5) Day 8: discard 100. Mu.L of liquid per well, and add liquid to supplement 50. Mu.L of serum-free medium/well containing 10ng/mLBMP4, 10ng/mL bFGF, 10ng/mLVEGF, 50ng/mL SCF and 10. Mu. Mol/L abscisic acid;

(6) Day 11: the inoculum was supplemented with 50. Mu.L of serum-free medium/well containing 10ng/mLBMP4, 10ng/mL bFGF, 10ng/mLVEGF, 50ng/mL SCF, 60ng/mL TPO and 10. Mu. Mol/L abscisic acid at a final TPO concentration of 20ng/mL;

(7) Day 14: collecting hematopoietic cells secreted by EB cells, transferring the cells to a 6-hole plate for culture, and continuously culturing for 5 days, wherein the culture solution is 2mL of serum-free culture medium/hole; the serum-free medium contains 20ng/mL SCF, 10ng/mL IL 11, 50ng/mL TPO and 10 mu mol/L abscisic acid;

(8) Day 19: megakaryocytes were collected.

5. The method of claim 4, wherein the differentiation efficiency of megakaryocytes in vitro is increased

Characterized in that the method also comprises the steps of reviving the human induced pluripotent stem cell and subculturing,

the cell recovery process comprises the following steps:

(1) Preheating the cell culture solution in a 37 ℃ water bath;

(2) Taking out the cell freezing tube from liquid nitrogen or-80 deg.C refrigerator, and shaking the freezing tube in 37 deg.C water bath to make it

Quickly thawing cells, quickly taking out the cells when Yu Xiao ice cubes exist in the liquid, transferring the cells to a super clean bench, adding 1mL of culture solution, uniformly mixing, and centrifuging at 1500rpm for 5 min;

(3) Discarding supernatant, adding preheated culture solution into the freezing tube, re-suspending cells, counting, seeding into 6-well plate,

mixing the cells uniformly by a cross method;

(4) 6-hole plateStanding at 37 deg.C and 5% CO ₂ Incubation in an incubator;

the subculture process comprises the following steps:

(1) Preparing a six-hole plate for bedding matrigel;

(2) Preheating a seashell culture solution in a water bath kettle at 37 ℃;

(3) Taking out the cell culture plate from the incubator, observing the growth state and density of the cell culture plate under a microscope, wherein the cell clone density reaches 70-80%, and the cell clone density can be passaged;

(4) Placing a culture dish of the human induced pluripotent stem cells in a super clean bench, removing a supernatant by using an aseptic pipette, taking 1mL of PBS, washing the cells once in the culture dish, and removing the PBS;

(5) Adding 1mL of 0.05mM EDTA into a culture dish, slightly shaking to fully pave the EDTA on the surface of cells, observing the edge of cell clone under a mirror to roll up, enabling the cells to become round, absorbing the EDTA by using a pipette gun, adding 2ml of Seebine culture solution by using the pipette gun, slightly blowing down the cells at the bottom of the culture dish, and carrying out passage to a 6-well plate paved with Matrigel at different densities according to the cell clone density;

(6) And observing the cell density after 24h, and carrying out next passage when the cell clone density reaches 70-80%.