A method and kit for inducing microglia from pluripotent stem cells

A technology of microglia cells and kits, applied in the field of stem cell biology, can solve the problems of few differentiation schemes, long time-consuming, difficult to control differentiation results, etc., and achieve the effect of reducing costs, reducing induction time, and simple methods

Active Publication Date: 2021-10-29
INST OF ZOOLOGY CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In addition, due to the late development of the microglia differentiation protocol and fewer differentiation protocols, the differentiation result is still difficult to control, and it takes a long time, even more than 60 days

Method used

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  • A method and kit for inducing microglia from pluripotent stem cells
  • A method and kit for inducing microglia from pluripotent stem cells
  • A method and kit for inducing microglia from pluripotent stem cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0153] Example 1 Method for Inducing Functional Microglia from Pluripotent Stem Cells

[0154] 1. Human induced pluripotent stem cells are induced into CD43+ hematopoietic progenitor cells, the process is as follows figure 1 Shown:

[0155] 1. Human induced pluripotent stem cells were cultured on a matrigel-coated six-well plate, using medium I (i.e. human pluripotent stem cell maintenance medium (PGM1 medium)), 37 ° C, 5% CO 2 Cultured in a humidified incubator until the cells adhered to 50%-70% (cultured for 4-5 days), during which the medium was changed once a day, 2 mL each time.

[0156] 2. Aspirate the supernatant and wash the six-well plate with 1×PBS, add 0.5mL ACCUTASE cell digestion solution to each well, 37°C, 5% CO 2 The humidified incubator was left to stand for 4 minutes, then 0.5 mL of PGM1 medium was added to terminate the digestion, and the cells were collected by centrifugation at 200 g for 5 minutes.

[0157] 3. Aspirate the supernatant and resuspend with...

Embodiment 2

[0172] Example 2 Co-culture experiment of microglial cells and mouse primary neurons

[0173] Isolate mouse primary neurons at 40,000 / cm 2 Insert into a 24-well plate with cell slides pre-coated with PDL, add 500 μL of neuronal medium, discard 200 μL of neuronal medium every two days and supplement with 300 μL of fresh neuronal medium.

[0174] After 14 days, mature microglial cells were inoculated at a ratio of 3:1, 250 μL of neuron medium and 250 μL of medium VIII were added, and the co-culture was continued for another three days. Aspirate the supernatant medium and add 500 μL of 4% paraformaldehyde solution to fix for 20 minutes. Rinse with 500 μL 1× phosphate buffered saline three times, 5 minutes each time, and discard the supernatant. Add 500 μL of blocking solution and block for 1 hour at room temperature. Aspirate and discard the blocking solution, add 200 μL of new blocking solution, add rabbit anti-human IBA1 antibody, mouse anti-human Nuclei antibody, goat anti-...

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Abstract

The invention discloses a method and a kit for inducing microglial cells from pluripotent stem cells. The present invention firstly provides a kit for inducing microglial cells. The present invention also provides a method for inducing microglial cells. The method and kit for inducing microglial cells of the present invention can successfully induce pluripotent stem cells into a large number of mature microglial cells with special morphology, expression of specific markers and good phagocytic function through primary hematopoietic progenitor cells. Co-cultivation technology and gene recombination technology, it only takes 35‑40 days to obtain mature microglia from pluripotent stem cells, reducing induction time and cost. The method is simple and convenient, making it possible to study the gene function of Key nodes in the development of glial cells and new therapeutic targets become possible, and also provide a basis for the construction of microglia with specific disease prevention and treatment functions, and provide a source for cell replacement therapy of microglia.

Description

technical field [0001] The invention relates to the field of stem cell biology, in particular to a method and a kit for inducing microglial cells from pluripotent stem cells. Background technique [0002] Stem cells (Stem Cells) are a type of insufficiently differentiated cells that exist in multicellular organisms. Stem cells can self-renew through mitosis to produce more stem cells, and can also divide and differentiate to form a variety of specialized cells, tissues and organs. Induced pluripotent stem cell (iPSC) is a new type of stem cell obtained by transferring transcription factors or small molecular compounds into mammalian somatic cells, and usually has three germ layers similar to embryonic stem cells Differentiation ability, in theory, it can differentiate into all tissues and organs of the adult. [0003] Induced pluripotent stem cells theoretically have the ability to differentiate into all types of specialized cells, and are very suitable for replacing cells...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/079
CPCC12N5/0622C12N2500/05C12N2501/115C12N2501/125C12N2501/145C12N2501/15C12N2501/155C12N2501/16C12N2501/165C12N2501/22C12N2501/2303C12N2501/2306C12N2501/2334C12N2501/727C12N2501/999C12N2506/1346C12N2506/30C12N2506/45
Inventor 滕兆乾李骞刘长梅杜洪震
Owner INST OF ZOOLOGY CHINESE ACAD OF SCI
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