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Stem cells for specifically expressing PD-1 as well as identifying and separating method and application of stem cells

A stem cell and specific technology, applied in the field of stem cells, can solve the problems of undiscovered, identified, and isolated stem cells with high stemness and lack of tissue-specific markers

Active Publication Date: 2018-03-23
北京泰盛生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, some substances that play a key role in regulating stem cell stemness have been found, but there is a lack of tissue-specific markers in the identification and isolation of stem cells with high stemness, especially the specificity that has a decisive regulatory effect on their biological activity and stemness Marker still not found

Method used

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  • Stem cells for specifically expressing PD-1 as well as identifying and separating method and application of stem cells
  • Stem cells for specifically expressing PD-1 as well as identifying and separating method and application of stem cells
  • Stem cells for specifically expressing PD-1 as well as identifying and separating method and application of stem cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0092] Example 1. Isolation and cultivation of stem cells from different sources

[0093] 1. Isolation and culture of SHED and DPSC

[0094] (1) Select and collect deciduous teeth and third molars of healthy subjects (requiring no history of periodontal disease and dental caries), rinse with PBS (phosphate buffered saline) buffer containing 100kU / L penicillin and 100mg / L streptomycin;

[0095] (2) The tooth is cut open, and the pulp tissue is extracted;

[0096] (3) Wash the pulp tissue with L-DMEM medium, centrifuge at 500-700g for 5 minutes, and discard the supernatant;

[0097] (4) Mix the tissue block and the medium according to the volume ratio of 2-3:1, inoculate them into a cell culture dish, and cultivate them in an incubator; the medium is a special serum-free medium for MSC;

[0098] (5) Change the medium every 3 days, and subculture when the cells reach about 80% fusion; the subculture medium is a serum-free medium specially used for MSC.

[0099] 2. Isolation ...

Embodiment 2

[0106] Example 2. Isolation and cultivation of stem cells from different sources

[0107] Mesenchymal stem cells are an important member of the stem cell family, derived from the mesoderm and ectoderm in the early stages of development, and exist in a variety of organs and tissues, including bone marrow, umbilical cord, adipose tissue, skeletal muscle, and oral cavity. Bone marrow stem cells (BMMSCs) are a type of mesenchymal stem cell often used for transplantation, mainly derived from bone marrow. Dental pulp tissue-derived mesenchymal stem cells (MSC-DP) is a neural crest-derived stem cell population with high proliferative ability, which can be isolated from the pulp of exfoliated deciduous teeth or permanent teeth.

[0108] 1. Human exfoliated deciduous tooth pulp stem cells (SHED) and adult dental pulp stem cells (DPSC)

[0109] (1) Collect the pulp tissues of people's retained incisors (deciduous teeth) and third molars (permanent teeth) respectively, and wash them r...

Embodiment 3

[0117] Example 3. Identification of osteogenic and adipogenic induction and odontogenic / osteogenic differentiation of stem cells from different sources

[0118] Osteogenic induction: 2 mM β-glycerophosphate (Sigma-Aldrich), 100 μM 2-phosphate ascorbic acid and 10 nM dexamethasone (Sigma-Aldrich) were added to the medium for osteogenic induction. After four weeks, mineralized nodule formation was observed by staining the cultures with mineralized alizarin red.

[0119] Microscopic observation showed that the cultured stem cells were successfully differentiated into osteoblasts.

[0120] Adipogenic induction: 500 nM xanthine (Sigma-Aldrich), 60 μM indomethacin (Sigma-Aldrich), 500 nM cortisol (Sigma-Aldrich), 10 μg / ml insulin (Sigma-Aldrich) and 100 μM 2-phosphate ascorbic acid Add to growth medium for adipogenic induction. One week later, positive cells were observed under a microscope by staining the cultured cells with Oil Red O (Sigma Aldrich).

[0121]Microscopic obser...

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Abstract

The invention discloses stem cells for specifically expressing a programmed death receptor 1 (PD-1), and particularly relates to the stem cells from an organic cavity. The stem cells comprise but notlimited to dental pulp stem cells, gingival mesenchymal steam cells (GMSCs), periodontal ligament stem cells (PDLSCs), stem cells of apical papilla (SCAPs) and dental follicle stem cells (DFSCs) or any combination thereof, and preferably comprise stem cells from human exfoliated deciduous teeth (SHED) and / or dental pulp mesenchymal stem cells for permanent teeth (DPSC); the stem cells also includemesenchymal stem cells from other tissues, which do not express the PD-1 at first and express the PD-1 after being modified by CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats), suchas PD-1 plus bone mesenchymal stem cells (BMMSC) modified by the CRISPR. The invention further discloses an identifying and separating method for the stem cells for specifically expressing the PD-1,a method for preparing the PD-1 plus mesenchymal stem cells modified by the CRISPR as well as application of the mesenchymal stem cells for expressing the PD-1, disclosed by the invention, in tissue regeneration, pain relief and treatment of chronic pain and a series of diseases.

Description

technical field [0001] The present invention relates to a stem cell specifically expressing programmed death receptor 1 (PD-1), especially a mesenchymal stem cell derived from oral tissue of a mammal (preferably human) specifically expressing PD-1. The invention also includes other mesenchymal stem cells that initially do not express PD-1 but are modified by CRISPR to express PD-1. The present invention also relates to a method for identifying and isolating stem cells specifically expressing PD-1, and preparing CRISPR-modified PD-1 + The method for mesenchymal stem cells, the PD-1-expressing mesenchymal stem cells of the present invention (including PD-1-expressing mesenchymal stem cells modified by CRISPR) are used for tissue regeneration, or pain relief / treatment, or treatment of immune-related diseases or inflammatory diseases or metabolic and degenerative diseases or some malignant tumors or fungal infections, and its application in the treatment of chronic pain and the p...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0775C12N5/10C12N15/85G01N33/68A61K35/28A61P35/00A61P29/00A61P31/10A61P37/02A61P3/00A61P21/04A61P19/02A61P19/04A61P19/08
CPCA61K35/28C07K14/70521C12N5/0662C12N15/85C12N2509/00C12N2510/00C12N2800/80C12N2810/10G01N33/68
Inventor 施松涛刘尧张建国张翔宇
Owner 北京泰盛生物科技有限公司
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