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Method for enhancing hematopoietic stem cell transplantation capacity

A technology of hematopoietic stem cells and ability, applied in the field of hematopoietic stem cells, can solve the problems of lack of HSC expansion scheme and low efficiency of HSC expansion

Active Publication Date: 2020-12-08
SUSHENG BIOTECH (HAINAN) CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to the low efficiency of SR1 on HSC expansion, UM171 has the potential to inhibit the erythroid differentiation of HSC, resulting in the lack of efficient and clear-cut HSC expansion schemes in clinical practice.

Method used

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  • Method for enhancing hematopoietic stem cell transplantation capacity
  • Method for enhancing hematopoietic stem cell transplantation capacity
  • Method for enhancing hematopoietic stem cell transplantation capacity

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Example 1 Separation of CD34 from umbilical cord blood + cell

[0027] 1) Collection of cord blood

[0028] The blood from the fetal umbilical cord was collected in a sterile environment in the operating room, stored in a blood bag with anticoagulant, temporarily stored in a microenvironment at 4°C, and delivered to the laboratory for use within 24 hours.

[0029] 2) Isolation of monocytes from cord blood

[0030] a) In a sterile laboratory bench, transfer the umbilical cord blood into a sterile culture bottle, add the corresponding volume of PBS according to the volume ratio of blood: phosphate buffer solution = 1:2, and mix well.

[0031] b) Slowly add the diluted umbilical cord blood into a 50ml centrifuge tube filled with 15ml of human lymphatic separation fluid. Be careful to add slowly to keep the interface between the two liquids clear and not to break the liquid level balance between blood and lymphatic separation fluid.

[0032] c) Centrifuge at 1500 rpm...

Embodiment 2

[0051] Example 2 CD34 + Cell culture and detection

[0052] experimental method:

[0053]a) Use 50-200ng / ml SCF (100ng / ml is preferred in this example), 50-200ng / ml Flt-3L (100ng / ml is preferred in this example), 10-100ng / ml TPO (50ng / ml is preferred in this example), 1 Resuspend CD34 in StemSpan medium with ~100μg / ml LDL (preferably 50μg / ml in this example) + For cells, add 8-20 μM (preferably 15 μM in this example) SP600125 / JNK-IN-8 / DMSO, add it to a 6-well low-attachment plate, and control the cell density not higher than 1×10 6 tablets / ml;

[0054] b) Place the cells at 37°C, 5% CO 2 cultured in a cell culture incubator;

[0055] c) Every 2 days, replace half of the medium to ensure a cell density of 1×10 6 grains / ml or less.

[0056] Cells were cultured for 48 hours, cellular RNA was extracted (QIAGEN RNAEASY PLUS MINI KIT cat:74136), RNA was reverse transcribed into cDNA, and qPCR experiments were carried out using cDNA as a template. The result is as figure 1 ,...

Embodiment 3

[0058] Embodiment 3CFU colony formation experiment

[0059] experimental method:

[0060] 1) Inoculate the cells obtained in Example 2 for 7 days into 2ml of methylcellulose, and mix well;

[0061] 2) Use a 1ml syringe to transfer the methylcellulose mixed with the cell suspension (about 10,000 cells) to a low-attachment 6-well plate;

[0062] 3) Place at 37°C, 5% CO 2 Cultivate in the concentration incubator for 14 days;

[0063] 4) Observe the number of different cell colonies formed under a microscope, and make a record.

[0064] Experimental results:

[0065] The result is as Figure 4 As shown, the results show that JNK inhibitors can significantly improve the colony-forming ability of hematopoietic stem cells.

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Abstract

The invention discloses a method for enhancing hematopoietic stem cell transplantation capacity. The hematopoietic stem cell transplantation capacity is enhanced by up-regulating expression of a hematopoietic stem cell CCN3 gene by adopting a JNK inhibitor. The method is clear in action mechanism, and the hematopoietic stem cells with high transplantation capacity can be effectively obtained, so that the problem that the number of the hematopoietic stem cells derived from the graft is insufficient is effectively solved.

Description

technical field [0001] The invention relates to the technical field of hematopoietic stem cells, in particular to a method for enhancing the ability of hematopoietic stem cell transplantation. Background technique [0002] Hematopoietic stem cells (HSC for short) are a type of adult stem cells with high self-renewal and multi-lineage differentiation potential, which can be used clinically to treat malignant and non-malignant blood diseases. However, due to the limited number of HSCs, most patients who urgently need HSC transplantation can only adopt conservative treatment strategies. In vitro expansion of HSC technology can solve the problem of insufficient cell number, and has a wide range of clinical application value. In recent years, significant progress has been made in the use of chemical small molecules to expand HSCs derived from cord blood in vitro. The two studies of the aryl hydrocarbon receptor inhibitor StemRegenin 1 (SR1) and the purine derivative UM171 both ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0789C12N5/02
CPCC12N5/0647C12N2501/125C12N2501/145C12N2501/26C12N2501/405C12N2501/998
Inventor 曾胜杨宗繁
Owner SUSHENG BIOTECH (HAINAN) CO LTD
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