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Method for separating and purifying adult ng2-positive stem cell population derived from multiple organs

A multi-organ, stem cell technology, applied in the field of isolation and purification of adult stem cell populations, can solve the problems of unclear NG2-positive cells, lack of separation and purification of NG2-positive cells, insufficient support for homology between PCs and NG2-positive cells, etc. Timely signal response, simple method and high purity effect

Active Publication Date: 2016-11-30
THE FIRST AFFILIATED HOSPITAL OF THIRD MILITARY MEDICAL UNIVERSITY OF PLA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there are currently many studies on NG2-positive cells as stem cells in the developing CNS, but due to the lack of practical methods for isolating and purifying NG2-positive cells from adult CNS, it is still unclear whether NG2-positive cells also have stem cell potential in adult CNS
In addition, recent studies have found that other organs other than the CNS also express NG2, such as vascular sinus pericytes (PCs), but the current research evidence is not enough to support the homology of PCs and NG2-positive cells

Method used

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  • Method for separating and purifying adult ng2-positive stem cell population derived from multiple organs
  • Method for separating and purifying adult ng2-positive stem cell population derived from multiple organs
  • Method for separating and purifying adult ng2-positive stem cell population derived from multiple organs

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Example 1. Separation and purification from adult mouse spinal cord NG2 positive stem cell population ( CNS-NG2 ）

[0028] Specific steps are as follows:

[0029] a. The spinal cords of C57BL / 6 adult mice weighing about 20 g and aged 6-9 weeks were isolated, the capsule was removed, and the tissues were thoroughly washed with MEM medium containing 100 U / mL penicillin and 100 µg / mL streptomycin and the tissue was broken;

[0030] b. Add 3 mL of trypsin / EDTA digestion solution to the disrupted tissue in step a, at 37°C, 5% CO 2 Incubate for 30 minutes under the same conditions, and then add 6 mL of DMEM / F12 medium containing 0.04% (w / w) DNase I, 10% (w / w) FBS, 100 U / mL penicillin and 100 µg / mL streptomycin, and pipet well. Disperse the cells, then filter through a cell sieve with a pore size of 70 µm to remove the tissue to obtain a single cell suspension, centrifuge at 2000 rpm for 5 minutes, and discard the supernatant;

[0031] c. Add 5 mL of 30% Percoll ...

Embodiment 2

[0036] Embodiment 2, adult body CNS-NG2 cell promotion EAE Functional fixes

[0037] Twenty female C57BL / 6 mice weighing 18-20g and aged 8-10 weeks were randomly divided into adult CNS-NG2 cell treatment group (EAE+CNS-NG2 group) and control group (EAE+PBS group). 10. The antigen MOG35-55 was diluted with physiological saline to make a solution of 6 mg / mL, and then mixed with an equal volume of complete Freund's adjuvant (the final concentration of Mycobacterium tuberculosis H37Ra was 4 mg / mL), emulsified, and 0.1 mL per mouse was added to the solution. The mice were injected subcutaneously at four points on both sides of the spine, and 0.5 mL of pertussis toxin (PTX) was intraperitoneally injected into the mice immediately after immunization (0 hour) and 48 hours to establish the EAE mouse model. The EAE mice were scored by a double-blind method once a day. The scoring criteria were as follows: 0: no clinical symptoms; 1: loss of tail tension with mild clumsiness; ...

Embodiment 3

[0040] Embodiment three, adult CNS-NG2 cell promotion SCI Functional fixes

[0041] The model of dorsal column entrapment spinal cord injury was constructed according to the literature method (Horn et al., 2008): use forceps with a width of 1.5mm to insert 1.0mm straight into the eighth thoracic vertebra of mice, close the tips of the forceps, and maintain the pressure for 10 seconds bell; repeat the above-mentioned crushing process twice, cover the wound with gel film, then suture the muscle layer with 4-0 nylon suture, and staple the entire wound with surgical staples.

[0042] (1) The adult CNS-NG2 cells obtained in Example 1 were injected every other day. Ten days later, it was observed that after the interaction between the adult CNS-NG2 cells and the SCI-injured neurons, they could stabilize and promote the growth of dystrophic neurites, and make them more stable. Protection from attack by inflammatory macrophages (this part of the research has been published: ...

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Abstract

The invention discloses an effective method for separating and purifying NG2-positive stem cell populations from multiple organs of an adult. Firstly, single cells in suspension are obtained by digesting and separating adult central nervous system, iris, bone marrow, heart, liver, pancreas, lung or kidney tissues NG2-positive stem cell populations were obtained by centrifugation of 30% and 70% percoll separation medium and then primary cultured. The obtained multi-organ-derived adult NG2-positive stem cell populations have high purity, strong proliferation ability, and similar developmental characteristics and stem cells. Potential, timely and strong response to injury signals, can quickly differentiate into functional repair cells in the corresponding organ injury microenvironment, and is expected to be used as a new type of stem cell population to effectively treat injured organs; The biological properties of cell populations and their role in regenerative medicine lay the foundation and provide novel stem cell populations for clinical stem cell therapy and regenerative medicine.

Description

technical field [0001] The invention belongs to the technical field of cell separation and purification, and relates to a method for separation and purification of adult stem cell populations. Background technique [0002] NG2 (Nerve / Glial 2) positive cells are a group of cells mainly distributed in the central nervous system (Central Nerve System, CNS), and are named Oligodendrocyte Procursor Cells (OPCs). According to the traditional concept definition, NG2 positive cells are the unique stem cell population with repair function in CNS. However, there are currently many studies on NG2-positive cells as stem cells in the developing CNS, but due to the lack of practical methods for isolating and purifying NG2-positive cells from adult CNS, it is still unclear whether NG2-positive cells also have stem cell potential in adult CNS . In addition, recent studies have found that other organs other than the CNS also express NG2, such as vascular sinus pericytes (PCs), but current ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/0797
Inventor 白莲花帅领赖洁娟曾林立张玉君李瑶琛张宏宇别平
Owner THE FIRST AFFILIATED HOSPITAL OF THIRD MILITARY MEDICAL UNIVERSITY OF PLA
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