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A method, composition and application for preparing pluripotent stem cell-like cells

A technology of pluripotent stem cells and compositions, applied in the field of stem cell repair, can solve the problems of stress, difficult pluripotent stem cells, and high risk of cell canceration, and achieve the effects of reducing costs, simple operation, and low risk of canceration

Active Publication Date: 2018-11-20
北京恩诺生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] (1) So far, it is still difficult to obtain a large number of pluripotent stem cells that meet the clinical treatment requirements at a reasonable cost in this field:
[0007] 1) The source of stem cells is limited: At present, pluripotent stem cells are mainly isolated from tissues and organs such as bone marrow, peripheral blood, liver and placenta, while the number of stem cells in bone marrow is only 1 / 10000, and the proportion in peripheral blood is only 1 / 10000. It is 1 / 250000, accounting for 1 / 100000 in the liver. Obtaining the amount of stem cells required for clinical treatment puts pressure on the donor body; although the number of stem cells derived from placenta is better than that of bone marrow, peripheral blood and liver, it is subject to ethical and medical concerns. Neoplastic limitations;
[0008] 2) Induced pluripotent stem cells (iPS) need to transfer Oct4, sox2, c-Myc, KLF4 and other genes into somatic cells, the operation method is complicated, the cost is high, and there are defects of high risk of cell canceration;
[0009] 3) Using a combination of M-CSF, IL-3, IL-6, IL-2 and anti-CD-13 antibodies to induce the formation of pluripotent stem cells, the preparation process requires the use of a combination of various cytokines or antibodies, and the cost High, and the efficiency of dedifferentiation of adult cells into pluripotent stem cells is low
[0010] (2) In terms of therapeutic effect, the repair ability of pluripotent stem cells to damaged or necrotic tissue, especially the damaged or necrotic tissue at the site of inflammation is poor, and the therapeutic effect is not ideal

Method used

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  • A method, composition and application for preparing pluripotent stem cell-like cells
  • A method, composition and application for preparing pluripotent stem cell-like cells
  • A method, composition and application for preparing pluripotent stem cell-like cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0108] Prepare peripheral blood-derived monocytes as follows:

[0109] 1) Collect peripheral blood samples. Take 4 sterile 50ml centrifuge tubes, carefully pour the samples into the centrifuge tubes, and put 50ml blood samples in each tube. 900g, centrifuge for 10min, slowly rise and fall slowly.

[0110] 2) Take the upper layer of yellow plasma into a new 50ml centrifuge tube (up to 3 / 4 of the serum layer to avoid loss of cell yield), and inactivate at 56°C for 15 minutes. Centrifuge at 3500rpm for 10min.

[0111] 3) Transfer the upper layer of plasma to a new 50ml centrifuge tube and store in a 4°C refrigerator for use.

[0112] 4) Resuspend the remaining blood sample to the original volume with PBS, prepare several sterile 50ml centrifuge tubes, add 20ml of human mononuclear cell separation solution to each tube, and do not splash the lymphocyte separation solution on the centrifuge tube wall (according to monocyte Separation solution: blood = 1:1, for example, 80ml who...

Embodiment 2

[0121] Drugs that induce dedifferentiation of monocytes to form pluripotent stem cell-like cells are screened in the following manner:

[0122] 1) Day 0: resuspend the monocytes prepared in Example 1, and make the density of the monocytes 1×10 6 / ml, inoculated into 24-well plates, at 37°C, 5% CO 2 Adhesive culture for 3 hours under the conditions; after that, the suspended cells were discarded, 0.5ml RPMI1640 medium was added to each well, and autologous serum was supplemented by 10%, and the cells were kept at 37°C and 5% CO 2 Continue to cultivate under the conditions of the medium, add M-CSF and autologous serum in the medium, and the final concentration of the M-CSF in the medium is 30ng / ml;

[0123] 2) Day 1, Day 2 and Day 3: Observe the cell morphology under the microscope every day.

[0124] Day3: Aspirate and discard 1 / 2 volume of medium in each well, and supplement 1 / 2 volume of fresh medium, which contains M-CSF (without adding the drug to be screened, as a contro...

Embodiment 3

[0134] Detect the ability of different types of Ophiopogon japonicus flavonoids to induce monocytes to form stem cell-like cells:

[0135] Refer to Example 2 for the specific method, the only difference is that the drugs to be tested are 6-formyl isotropis flavanone A, methyl radix flavanone B, methyl philopogon flavanone A, 6-formyl Isoflavone A and 6-formyl isoflavone B, see Table 2 for specific test results. According to the data shown in Table 2, it can be seen that the above five radix flavonoids can inhibit the expression of CD14 gene and up-regulate the expression of CD90 gene, and have the ability to induce monocytes to form stem cell-like cells. Among them, 6-formyl isoflavone A has the strongest inductive ability.

[0136] Table 2 The ability of different Ophiopogon japonicus flavonoids to induce monocytes to form stem cell-like cells

[0137]

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Abstract

The invention relates to a preparation method of a multipotential stem cell-like cell, a composition and an application. The method includes steps of 1), culturing a mononuclear cell in a culture mediu containing a cell growth factor M-CSF, wherein the working concentration of the M-CSF in the culture medium is 10-50 ng / ml, and the culture time is 3-5 days; 2), after step 1), culturing the mononuclear cell in the culture medium containing M-CSF and ophiopogonone to obtain the multipotential stem cell-like cell, wherein the working concentrations of the M-CSF and ophiopogonone in the culture medium are 10-30 ng / ml and 30-100 mu g / ml; the culture time is 4-8 days. Compared with the traditional stem cell induction method, the method has the advantages of being simple in operation, high in inducing efficiency and high in performance cost.

Description

technical field [0001] The present invention relates to the field of stem cell repair, in particular to a method, composition and application for preparing pluripotent stem cell-like cells. Background technique [0002] Stem cells are undifferentiated cells before the differentiation stage. According to different differentiation capabilities, they can be roughly divided into totipotent stem cells, pluripotent stem cells and monopotent stem cells. Among them, totipotent stem cells refer to all cells from the fertilized egg to 32 cells in the cleavage stage, which have the potential to form a complete individual differentiation; while pluripotent stem cells have the ability to differentiate into various cells, and can form various cells and organs, but cannot Form a complete individual; while unipotent stem cells, also known as multipotent stem cells or partial stem cells, can only differentiate into one type or two closely related types of cells. [0003] Totipotent stem cel...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/078C12N5/074A61K35/545A61K45/06A61P19/02A61P29/00A61P9/10A61P25/00A61P37/02A61P1/16A61P13/12A61P3/10A61P3/00A61P35/00A61K35/15
CPCA61K35/15A61K35/545A61K45/06C12N5/0645C12N5/0696C12N2500/30C12N2501/22A61K2300/00
Inventor 肖敏张倩王建开邵进士
Owner 北京恩诺生物科技有限公司
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