Application of JAK (Janus kinase) in preparation of products for promoting induction of multipotent stem cells to generate megakaryocytes and platelets

A pluripotent stem cell and kinase inhibitor technology, applied in artificially induced pluripotent cells, embryonic cells, animal cells, etc., can solve the problems of poor operability, low yield, long cycle, etc., and achieve short differentiation cycle and yield. High and simple operation effect

Inactive Publication Date: 2019-10-25
INST OF HEMATOLOGY & BLOOD DISEASES HOSPITAL CHINESE ACADEMY OF MEDICAL SCI & PEKING UNION MEDICAL COLLEGE
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] One aspect of the present invention is aimed at the disadvantages of poor operability, long cycle and low yield in the method of inducing megakaryocytes and platelets from pluripotent stem cells in the prior art, and provides a JAK kinase inhibitor in the preparation of promoting pluripotent stem cells. Use in products that induce the production of megakaryocytes and platelets

Method used

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  • Application of JAK (Janus kinase) in preparation of products for promoting induction of multipotent stem cells to generate megakaryocytes and platelets
  • Application of JAK (Janus kinase) in preparation of products for promoting induction of multipotent stem cells to generate megakaryocytes and platelets
  • Application of JAK (Janus kinase) in preparation of products for promoting induction of multipotent stem cells to generate megakaryocytes and platelets

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0067] Example 1: Preparation of megakaryocytes and platelets

[0068] 1) Preparation of human pluripotent stem cells

[0069] a. According to the human pluripotent stem cell single-cell subculture procedure, digest human pluripotent stem cells with Accutase for 3-5 minutes. After the clones are loose, add DMEM / F12 and pipette with a 1mL gun tip to form single cells.

[0070] b. Centrifuge at 300 g for 5 min at room temperature, and discard the supernatant.

[0071] c. Resuspend the cells in mTeSR medium added with Y-27632 (final concentration: 5 μM), and add 3.5×10 5 Growth Factor Reduced Matrigel (Growth Factor Reduced Matrigel)-coated Petri dishes were seeded at cell densities per 10 cm dish.

[0072] d. After cross-shaking, place at 37°C, 5% CO 2 Incubate for 24 hours in the incubator.

[0073] 2) Hematopoietic differentiation of human pluripotent stem cells

[0074] a. After 24 hours, add Activin A (Activin A, final concentration of 50 ng / mL), bone morphogenetic prot...

Embodiment 2

[0087] Example 2: Detection of producing megakaryocytes

[0088] The DMSO control group and the JAK kinase inhibitor group were respectively set up.

[0089] a. Collect about 1×10 cells on the third day of megakaryotic differentiation 5 The cell suspension was centrifuged at 300 g for 5 min, and the cell pellet was resuspended with 100 μL of 0.2% BSA.

[0090] b. Add 1 μL anti-CD41a-APC (BD) and 1 μL anti-CD42b-PE (BD) flow antibody to each group, incubate in the dark for 30 minutes, and detect CD41a by flow cytometry (FACS Canto II; BD Biosciences) + CD42b + The proportion of megakaryocytes.

[0091] c. The result is as follows figure 2 As shown, on the 3rd day of megakaryotic induced differentiation, the control group (DMSO group) CD41a + CD42b + The proportion of megakaryocytes is over 70%. After treatment with JAK kinase inhibitors, the purity of megakaryocytes can be as high as 90%.

Embodiment 3

[0092] Example 3: Detection of produced platelet particles

[0093] The DMSO control group and the JAK kinase inhibitor group were respectively set up.

[0094] a. Collect the cell suspension on the sixth day of megakaryotic induction, centrifuge at 300 g for 5 min, and take 200 μL of supernatant.

[0095] b. Add 1 μL anti-CD41a-APC (BD) and 1 μL anti-CD42b-PE (BD) flow-type antibodies to each group, incubate in the dark for 30 minutes, and run the machine (FACS Canto II; BD Biosciences) for detection.

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Abstract

The invention discloses application of JAK (Janus kinase) in the preparation of products for promoting induction of multipotent stem cells to generate megakaryocytes and platelets. A megakaryocytic differentiation system established herein has higher operability and repeatability and can provide efficient rapid in-vitro generation of active platelets. Rapid efficient in-vitro generation of platelets is achieved under serum-free stromal cell-free culture conditions; specific functional micromolecules are added in the megakaryocytic differentiation stage through small-scale molecular screening,and the ratio of active platelets (CD41a<+>CD42b<+>) is further increased. Statistical results show that the generation of 27-36 CD41a<+>CD42b<+> active platelets can be induced through single human polypotent stem cells, and the results are evidently higher than the results of the prior art. The rapid simple serum-free stromal-free culture strategy lays the basis for the large-scale production offunctional platelets for clinical therapy.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to the use of JAK kinase inhibitors in the preparation of products for promoting the induction of pluripotent stem cells to produce megakaryocytes and platelets. Background technique [0002] As the smallest blood cell in mammalian blood, platelets play an important role in physiological and pathological processes such as hemostasis, wound healing, inflammatory response, thrombosis, and organ transplant rejection. Donor-derived platelet concentrate transfusions are the only means of treatment for severe blood loss, anemia, thrombocytopenia, and thrombocytopenia due to radiation and chemotherapy. Due to the limited source of donors, short in vitro storage period (4-6 days), pathogenic contamination, and ineffective transfusion, the contradiction between supply and demand of platelets has become increasingly acute, which greatly limits its clinical application (Yin YH, Li CQ, Liu Z. Bloo...

Claims

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Application Information

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IPC IPC(8): C12N5/0786C12N5/078A61K35/19A61K35/28A61P7/06A61P7/04A61P1/16
CPCA61K35/19A61K35/28A61P1/16A61P7/04A61P7/06C12N5/0644C12N5/0645C12N2501/115C12N2501/125C12N2501/145C12N2501/15C12N2501/155C12N2501/16C12N2501/165C12N2501/2303C12N2501/2306C12N2501/2311C12N2501/405C12N2506/02C12N2506/45C12N2533/54
Inventor 周家喜刘翠翠刘懿莹苏培王洪涛
Owner INST OF HEMATOLOGY & BLOOD DISEASES HOSPITAL CHINESE ACADEMY OF MEDICAL SCI & PEKING UNION MEDICAL COLLEGE
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