Application ofhistone deacetylase inhibitor in preparation of products for promoting differentiation of pluripotent stem cells into hematopoietic stem and progenitor cells

A sirtuin and pluripotent stem cell technology, applied to artificially induced pluripotent cells, embryonic cells, animal cells, etc., can solve the problem of low yield of hematopoietic stem and progenitor cells, achieve short processing time, rapid production, and reliable Operable and reproducible effects

Active Publication Date: 2019-10-25
天津协和生物科技开发有限公司 +2
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0006] One aspect of the present invention is to solve the problem of low yield of hematopoietic stem and progenitor cells induced by small molecules in the prior art, and provides histone deacetylase inhibitors to promote the differentiation of pluripotent stem cells into hematopoietic stem and progenitor cells. Use in the product

Method used

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  • Application ofhistone deacetylase inhibitor in preparation of products for promoting differentiation of pluripotent stem cells into hematopoietic stem and progenitor cells
  • Application ofhistone deacetylase inhibitor in preparation of products for promoting differentiation of pluripotent stem cells into hematopoietic stem and progenitor cells
  • Application ofhistone deacetylase inhibitor in preparation of products for promoting differentiation of pluripotent stem cells into hematopoietic stem and progenitor cells

Examples

Experimental program
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Effect test

Embodiment 1

[0042] Example 1: Culture and passage of human pluripotent stem cells

[0043] Human pluripotent stem cells (human embryonic stem cell line H1) utilize mTeSR TM 1 (Stem Cell Technologies) medium was cultured on cell culture plates covered with Mrigel (corning) matrigel to maintain its self-renewal.

[0044] Cloning passage: Add 500 μl Dispase enzyme (Stem Cell Technologies) to digest the larger clones in the 12-well plate for 5 minutes, aspirate the enzyme solution and add 1ml DMEM / F12 (Hyclone) medium, blow down the clones and put them into a 15ml centrifuge tube , room temperature, centrifuge at 350g for 5min. Discard the supernatant and add mTeSR TM 1 Resuspend the pellet, adjust the density and transfer it to a 12-well plate of cells covered with Mrigel Matrigel, shake well, and store at 37°C, 5% CO 2 cultured in an incubator.

[0045] Single cell passage: Add 500 μl Accutase enzyme (Gibco) to the clones that grow larger in the 12-well plate, digest at 37°C for 3 minut...

Embodiment 2

[0046] Example 2: Hematopoietic differentiation of human pluripotent stem cells

[0047] 1) Human pluripotent stem cell lines were digested into single cells, and after adding Rho kinase inhibitor Y-27632, 3.5×10 4 The density of single cells / well was subcultured on a 12-well plate covered with Growth Factor Reduced Mrigel (corning), shaken evenly, and kept at 37°C, 5% CO 2 cultured in an incubator.

[0048] 2) After 24 hours, the culture medium was discarded, and Custom mTeSR1 (StemCell Technologies) medium containing 40 ng / ml ActivinA (Peprotech), 50 ng / ml BMP4 (Peprotech) and 100 μm Vorinostat was added to induce differentiation for 2 days.

[0049] 3) Discard the medium, add CustommTeSR1 containing 40ng / ml VEGF (Peprotech) and 50ng / ml bFGF (Peprotech) to continue to induce differentiation for 2 days, and replace fresh medium every 24h.

[0050] 4) Discard the medium, add Custom mTeSR1 containing 40 ng / ml VEGF, 50 ng / ml bFGF and 20 μM SB 431542 (STEMGENT) to continue cult...

experiment example 1

[0051] Experimental Example 1: Flow Cytometry Detection of Hematopoietic Stem and Progenitor Cells

[0052] 1) Add the last 500 μl of Accutase enzyme to the cells cultured for 8 days, let stand for digestion at 37°C for 5 minutes, resuspend in 1ml DMEM / F12, pour into 1.5ml EP tube, centrifuge at 350g for 5min at room temperature.

[0053] 2) Discard the supernatant, resuspend each group of samples with 100 μl 0.2% BSA, add 0.5 μl anti-CD31-APC and anti-CD43-PE, and incubate on a horizontal shaker for 30 minutes in the dark, then put on the machine (FACS Canto II; BD Biosciences) detection.

[0054] 3) IgG was used as the negative control gate, and flow cytometry results were analyzed.

[0055] 4) Results such as figure 1 showed that early addition of HDAC inhibitors effectively increased CD31 compared with DMSO controls + CD43 + The proportion of hematopoietic stem and progenitor cells.

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Abstract

The invention discloses application of a histone deacetylase inhibitor in preparation of products for promoting differentiation of pluripotent stem cells into hematopoietic stem and progenitor cells.According to the application, differentiation of pluripotent stem cells of every 104 personscan be promoted to produce 15.7% of CD31+CD43+hematopoietic stem and progenitor cells, which are nearly 4 times more than those ofa control group, the generation efficiency of the hematopoietic stem and progenitor cells is also significantly higher than thatof thehematopoietic stem and progenitor cellsof the general range,generatedafter promotion by other small molecules in the prior art, the treatment time is short, the maneuverability and the repeatability are higher, and the hematopoietic stem and progenitor cells can be efficiently and rapidly generated in vitro. According to the application of the histone deacetylase inhibitor in preparation of the products for promoting differentiation of thepluripotent stem cells into the hematopoietic stem and progenitor cells, by adding the officinal HDAC inhibitor at the early stage of differentiation, the serum-free and matrix-free mode of highly-controllably and efficiently generating the hematopoietic stem and progenitor cells at a low costlays a foundation for large-scale production of functional blood cells for clinical treatment in the future.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to the use of histone deacetylase inhibitors in the preparation of products for promoting the differentiation of pluripotent stem cells into hematopoietic stem progenitor cells. Background technique [0002] "Blood supply shortage" (blood supply shortage) refers to the phenomenon of partial blood type or emergency. In recent years, among the 70 large and medium-sized cities in the Mainland, as many as 40 "blood shortages" have been reported. Direct transfusion of blood cells is an effective means to alleviate ischemia in patients with blood diseases and traumatic bleeding. However, domestic "blood shortage", blood product contamination, immune rejection and other problems lead to insufficient blood sources, which brings troubles to the treatment of blood diseases. Therefore, the large-scale production of functional blood cells in vitro from autologous stem cells without immune rejectio...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0789A61K35/28A61P7/06A61P7/00
CPCA61K35/28A61P7/00A61P7/06C12N5/0647C12N2501/065C12N2501/115C12N2501/15C12N2501/155C12N2501/16C12N2501/165C12N2501/405C12N2506/02C12N2506/45C12N2533/54
Inventor 周家喜温玉琪王洪涛刘翠翠苏培
Owner 天津协和生物科技开发有限公司
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