Methods of expanding hematopoietic stem cells and compositions thereof

A technology of hematopoietic stem cells and compositions, which is applied in the field of hematopoietic stem cell culture and expansion, can solve problems such as insufficient HSCs that cannot be effectively solved, and achieve the effects of maintaining stemness, solving insufficient sources, and expanding HSCs

Pending Publication Date: 2022-02-22
EDIGENE GUANGZHOU INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The problem of insufficient HSCs faced by

Method used

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  • Methods of expanding hematopoietic stem cells and compositions thereof
  • Methods of expanding hematopoietic stem cells and compositions thereof
  • Methods of expanding hematopoietic stem cells and compositions thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0097] Example 1: Sorting CD34+HSC from umbilical cord blood for subsequent small molecule screening

[0098]Prepare reagent H-lyse Buffer (1×) solution and Wash Buffer (1×) solution. Take 5ml of H-lyseBuffer 10× stock solution (R&D, product number: WL1000), add 45ml of deionized water (filtered through a 0.22μm membrane), mix well, and prepare H-lyse Buffer (1×) solution. Take 5ml of Wash Buffer 10× stock solution (R&D, product number: WL1000), add 45ml of deionized water, mix well, and prepare Wash Buffer (1×) solution.

[0099] Add physiological saline to 10ml of fresh human cord blood to a final volume of 30ml. Add human lymphocyte separation medium (Daktronics, product number: DKW-KLSH-0100) to the diluted blood, then centrifuge at 400g for 30min (set speed up to 3, speed down to 0), absorb the buffy coat, and centrifuge at 500g for 10min. Concentrate the cell pellet into a 50ml centrifuge tube, add 10ml of H-lyse Buffer (1×), and lyse the red blood cells at room temper...

Embodiment 2

[0101] Example 2: Culture and preparation of HSC cells

[0102] According to the solubility indicated in the instructions of the compound and the required solvent (see Table 1 for the product number of the compound), the mother solution was prepared, and the following medium was prepared according to the set concentration gradient: SFEMII medium (stem cell, product number: 09655)+50ng / ml Flt-3L (PeProtech, Cat. No.: 300-100UG) + 50ng / ml SCF (PeProtech, Cat. No.: 300-07-100UG) + 50ng / ml TPO (PeProtech, Cat. No.: 300-18-100UG) + 10ng / ml IL -6 (PeProtech, product number: 200-06-20UG) + 1% penicillin-streptomycin double antibody (HyClone, product number: sv30010).

[0103] First, the prepared medium was added to a 24-well plate (Corning, product number: 3473), 950 μL per well, and placed in a carbon dioxide incubator (Thermo, model: 3111) to preheat; the spare HSC in Example 1 Resuspend with SFEMII+50ng / mlFlt-3L+50ng / ml SCF+50ng / ml TPO+10ng / ml IL-6+1% double antibody, according ...

Embodiment 3

[0108] Example 3: Establishment of a detection method for flow cytometric detection of HSC stemness

[0109] The antibodies used in this example and their sources are shown in Table 2.

[0110] Table 2: Antibodies

[0111]

[0112] Sample 20 μL of cells cultured to 6-8 days (D6-D8) in the above-mentioned Example 2 and count them, and take out a suspension of 2*10^5 cells into a 1.5 ml centrifuge tube according to the counting results; centrifuge at 400 g for 5 min, discard supernatant. Take 100 μL of PBS (phosphate-buffered saline, HyClone, product number: SH30256.01) containing 1% HSA (human serum albumin, Guangdong Shuanglin, product number: S10970069), resuspend the cells, vortex to mix, and set aside. Then, the cells cultured under the same conditions but not treated with the compound were collected according to the same method as the sample cells to be tested, and divided into 4 groups, including: negative control (NC) group, isotype control (ISO) group, APC fluoresc...

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Abstract

The invention provides a composition for amplifying hematopoietic stem cells and/or prolonging the stemness retention time of the hematopoietic stem cells, which comprises a cell culture medium and heparinase, and provides a kit comprising the composition. The invention further relates to a method for expanding the hematopoietic stem cells and/or prolonging the stemness retention time of the hematopoietic stem cells by using the composition, the hematopoietic stem cells cultured by using the method, and a method for treating diseases by using the hematopoietic stem cells. And a new solution is provided for amplifying the hematopoietic stem cells and/or prolonging the stemness retention time of the hematopoietic stem cells.

Description

technical field [0001] This application relates to the field of hematopoietic stem cell culture and expansion, as well as therapeutic products and disease therapies related to hematopoietic stem cell transplantation. Background technique [0002] Hematopoietic stem cells (HSCs) are a group of heterogeneous primitive hematopoietic cells in the blood system, which have two important characteristics of self-renewal and multi-lineage differentiation. Hematopoietic stem cells isolated from bone marrow, mobilized peripheral blood cells, umbilical cord blood, fetal hematopoietic system, embryonic stem cells, etc. usually contain: long-term hematopoietic stem cells (LT-HSC, which can maintain self-renewal and multi-lineage differentiation potential throughout life); short-term hematopoietic stem cells (ST - HSCs, with multi-lineage differentiation capacity but limited self-renewal potential, typically repopulating the myeloid and / or lymphoid lineage at short intervals, about six wee...

Claims

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Application Information

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IPC IPC(8): C12N5/0789A61K35/28A61P37/06A61P37/02A61P7/00A61P35/02A61P35/00A61P31/18
CPCC12N5/0647A61K35/28A61P37/06A61P37/02A61P7/00A61P35/02A61P35/00A61P31/18C12N2501/73C12N2501/065
Inventor 方日国史忠玉杨卉慧袁鹏飞
Owner EDIGENE GUANGZHOU INC
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