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Method for differentiating human induced pluripotent stem cells into peripheral neurons

A technology of pluripotent stem cells and peripheral nerves, applied in the field of differentiation from human induced pluripotent stem cells to peripheral neurons, can solve the problems of short induction time, differentiation efficiency, unclear culture conditions, and high state requirements, so as to avoid the risk of pathogenic infection, Effect of improving differentiation efficiency and improving safety

Pending Publication Date: 2021-12-21
HELP STEM CELL INNOVATIONS CO LTD
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  • Abstract
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  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Although hiPSCs can be successfully differentiated into peripheral neurons by various methods, the co-culture method of stromal cells was mainly used in the early stage. Most of the stromal cells were PA6 cells. Due to the existence of stromal cells, the culture conditions were unclear and the induction time was relatively long. Long and low differentiation efficiency
In addition, researchers have been working on optimizing the combination of small molecule inhibitors in order to obtain peripheral neurons with short induction time and high differentiation efficiency, but the purity of peripheral neurons differentiated by these methods still needs to be further improved, and the initial The state of iPSC has high requirements, depends on co-cultured cells in the feeder layer, and there are uncertain culture factors (such as serum, conditioned medium, etc.)

Method used

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  • Method for differentiating human induced pluripotent stem cells into peripheral neurons
  • Method for differentiating human induced pluripotent stem cells into peripheral neurons
  • Method for differentiating human induced pluripotent stem cells into peripheral neurons

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Embodiment 1, induction differentiation period

[0040] The iPSCs were digested into a single-cell suspension, and peripheral neuron differentiation medium P was added to terminate the digestion. The day of resurfacing induction was recorded as D0', and then fresh peripheral neuron differentiation medium P was completely replaced every day from day D2', and Observe the morphology of hiPSCs and culture them for 6-8 days until D8'-D10'.

[0041] Steps to induce differentiation in the induction preparation phase include:

[0042] S1.1: Digest the stem cells with a polymerization degree of 70-90% into a single cell suspension, and add an equal volume of peripheral neuron differentiation medium P to terminate the digestion;

[0043] S1.2: Divide the cells at a rate of 1 cm 2 Contains 0.1-5×10 4 The density of cells was inoculated in a culture flask pre-coated with Matrigel glue, shaken to distribute the cells evenly, and then placed in an incubator, and the day of resurfa...

Embodiment 2

[0052] Embodiment 2, induction maturation period

[0053] Induced maturation stage of step 2: the day of resurfacing maturation is recorded as D0, and the peripheral neuron differentiation medium P in the culture flask of step 1 is replaced with peripheral neuron maturation medium N1 or peripheral nerve maturation medium N2, and cultivated to D10.

[0054] The D0 recorded on the day of resurfacing maturation in the induction maturation stage is the retiming after D8' or D10' of the induction preparation stage.

[0055] The steps to change the medium during the induced maturation phase include:

[0056] S2.1: Peripheral neurons enter the mature stage, digest the cells and divide each 1cm 2 Including (0.5~5)×10 4 The density of cells was inoculated in the plate coated with Matrigel glue in advance, and the peripheral neuron differentiation medium P in the culture flask was replaced with peripheral neuron maturation medium N1, and D0 was used on the day of resurfacing maturatio...

Embodiment 3

[0064] Embodiment 3, cell immunofluorescence (Confocal)

[0065] The neuron cells obtained after the operations in Example 1 and Example 2 were stained by the neuron-specific marker Tuj1 and the peripheral neuron-specific marker Brn3a, respectively. see results figure 1 and figure 2 ;

[0066] figure 1 It is the result of immunofluorescence staining of the neuron marker Tuj1, in which the green fluorescent cells indicate TUJ1 positive, and TUJ1 positive indicates that the cells are neurons; figure 2 It is the result of immunofluorescence staining of the mature neuron marker Brn3a, in which the light green fluorescent cells indicate that the neurons are Brn3a positive, and the Brn3a positive indicates that the cells are mature peripheral neuron cells.

[0067] Identification methods for neuronal (overall) and peripheral neuron purity:

[0068] Use 4% paraformaldehyde as a solvent to prepare a fixed-block permeabilization solution containing 1% bovine serum albumin (BSA) ...

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Abstract

The invention discloses a method for differentiating human induced pluripotent stem cells into peripheral neurons and belongs to the field of stem cells in neuroscience. The method comprises an induction preparation stage and an induction maturation stage, and the induced pluripotent stem cells can be differentiated into the neurons in vitro. The method is short in induction time and high in purity. Furthermore, the method can be used for replacing clinical treatment of neurodegenerative diseases by stem cell transplantation, such as treatment of Parkinson's disease, spinal cord injury, glial cell related diseases and the like, and has a wide application prospect.

Description

technical field [0001] The invention relates to the field of neuroscience stem cells, in particular to a method for differentiating human induced pluripotent stem cells into peripheral neurons. Background technique [0002] Induced peripheral nerves refer to all nerves other than the brain and spinal cord, including ganglia, nerve trunks, nerve plexuses, and nerve terminal devices; drugs for the peripheral nervous system include cholinergic drugs, adrenergic receptor agonists, and histamine receptor antagonists Drugs, local anesthetics, etc., the efficacy and toxicity of such drugs can be studied through in vitro cell models of peripheral neurons. In addition, many drugs can cause peripheral nervous system dysfunction, and suitable in vitro cytotoxicity models are urgently needed for research testing. At present, drug-induced neurotoxicity is mainly studied with rodent in vitro models. However, this model has certain limitations. The reliability of extrapolating rodent test...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0793
CPCC12N5/0619C12N2506/45C12N2501/999
Inventor 夏伟荣徐轶冰黄琼陈涛涛王嘉显
Owner HELP STEM CELL INNOVATIONS CO LTD