Universal human-derived stem cell suitable for allograft and construction method thereof

A stem cell and cell technology, applied in the medical field, can solve problems such as immune rejection, achieve low immunogenicity, and avoid the effect of allogeneic immune response

Active Publication Date: 2021-12-28
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

It is expected that it can be transplanted into the patient without considering HLA matching and can avoid being attacked by the patient's immune system, and play

Method used

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  • Universal human-derived stem cell suitable for allograft and construction method thereof
  • Universal human-derived stem cell suitable for allograft and construction method thereof
  • Universal human-derived stem cell suitable for allograft and construction method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0093] Example 1: IFN-γ Up-regulates HLA-I Gene Expression of MSCs

[0094] This example proves through specific experiments that IFN-γ can up-regulate the expression of the HLA-I gene of MSCs.

[0095] 1.1 Cell culture and treatment

[0096] MSC culture conditions: L-DMEM medium (containing 10% fetal bovine serum), 5% CO2, 37°C constant temperature culture. 24 hours before treatment, MSCs were treated with 5 × 10 5 Each well was seeded into a 6-well plate for culture, and the cell confluency reached 60%-70% the next day.

[0097] The cultured MSCs were divided into three groups, the first group was blank control, the second group added IFN-γ to the medium at a concentration of 100ng / ml, and continued to culture for 24 hours; the third group, IFN-γ was added at a concentration of 100ng / ml -γ was added to the culture medium and continued to culture for 48 hours; the cells were collected for subsequent processing such as RNA extraction.

[0098] 1.2 MSC RNA extraction and fl...

Embodiment 2

[0122] Example 2: Experiments involving the participation of enhancers in the process of IFN-γ stimulating HLA-I expression

[0123] In this example, JQ1 (purchased from: abcam; product number: ab146612; batch number: APN15092-1-1; an enhancer inhibitor) with three concentrations of 125, 250 and 500nmol / ml were used to pretreat MSCs for 2 hours, and then Add 100ng / ml IFN-γ to treat MSC, continue to culture for 48 hours, collect cells and extract RNA for qPCR to detect the expression of HLA-I related genes, and set the corresponding MSC blank control, and MSC blank control plus IFN-γ treatment, A total of five groups of MSCs were compared and detected by q-PCR and flow cytometry respectively. The results are shown in Figure 3. For the specific process and sequence of q-PCR, refer to the process and conditions of Example 1. For the q-PCR results, see Figure 3A , is the expression level of HLA-A, HLA-B, HLA-C and B2M of MSC in the five groups; the results of flow cytometry are s...

Embodiment 3

[0136] Example 3, IFN-γ has a promoting effect on the opening and activation of the enhancer near the B2M gene

[0137] In order to further understand whether there are enhancers involved in the vicinity of HLA-I related genes after IFN-γ stimulation, and whether it is directly related to the expression of that gene, we performed H3K27ac ChIP-Seq analysis and bioinformatics analysis.

[0138] 1. The process of H3K27ac ChIP-Seq analysis is as follows:

[0139] 1 x 10 at room temperature 7 The non-IFN-γ-stimulated MSCs and IFN-γ-stimulated MSCs were respectively fixed with 1% formaldehyde for 10 minutes to obtain DNA-protein crosslinks. After sonicating for 1.5 hours to obtain 200-300bp chromatin fragments, mix with H3K27ac antibody (H3K27ac is a recognized active enhancer marker protein, and use H3K27ac antibody to capture all enhancer DNA bound to H3K27ac protein) at 4°C After overnight incubation, antibody-chromatin complexes were obtained by incubating with Dynabeads Prote...

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Abstract

The invention provides a universal human-derived stem cell suitable for allograft and a construction method thereof. By knocking down or silencing an enhancer gene sequence part of a B2M gene in the stem cell, the stem cell can maintain low immunogenicity in an inflammatory environment and desensitize inflammatory factor IFN-gamma stimulation; and the immune response of allogeneic peripheral blood mononuclear cells is not caused, and meanwhile, the killing effect of allogeneic CD8+T cells and NK cells can be avoided. It is expected that under the condition that HLA matching is not considered, the stem cell can be transplanted into the body of a patient and can be prevented from being attacked by an immune system of the patient, the treatment effect is achieved, and the problem of immunological rejection of the human immune system to allogeneic source cells in a regeneration therapy based on stem cell transplantation is solved.

Description

technical field [0001] The present invention firstly belongs to the field of medicine, specifically relates to a kind of universal human stem cell, which can be used for allogeneic transplantation without causing rejection; secondly belongs to the field of biotechnology, specifically relates to a method for constructing universal human stem cell. Background technique [0002] The description of the background technology is only a general description for the convenience of understanding the content of the present invention, and does not constitute any limitation to the present invention. [0003] The occurrence of transplant rejection mainly depends on the histocompatibility between the donor and the recipient, among which the matching degree of the major histocompatibility antigen (MHC) molecules is particularly important. Human MHC molecules, also known as human leukocyte antigens (HLA), are highly polymorphic. Since the HLA molecular types between donors and recipients are...

Claims

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Application Information

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IPC IPC(8): C12N5/10C12N15/113C12N9/22C12N15/867
CPCC12N5/0662C12N5/0667C12N15/113C12N9/22C12N15/86C07K14/70539C12N2310/20C12N2510/00C12N2740/15043
Inventor 柳华王飞
Owner ZHEJIANG UNIV
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