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A preparation method and application of oligodendrocytes

A technology of oligodendrocytes and pluripotent stem cells, applied in the field of stem cell therapy, can solve the problems of time-consuming, low efficiency or safety

Active Publication Date: 2022-06-24
SHENZHEN EXONEUGLIA BIOMEDICAL TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, most current methods for generating OPCs are time-consuming or based on lentiviral technology, which suffer from low efficiency or safety issues.

Method used

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  • A preparation method and application of oligodendrocytes
  • A preparation method and application of oligodendrocytes
  • A preparation method and application of oligodendrocytes

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0113] 1.1 Cell Culture and Cell Differentiation

[0114] Human induced pluripotent stem cell (hiPSC) line (UE017C1) derived from Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences was cultured on Matrigel-coated six-well plates in a 37°C incubator using mTeSR™ Base (Stem Cell Technologies, Canada), CO 2 for a 5% incubator.

[0115]To generate PAX6+ / NESTIN+ / SOX2+ NSCs, a dual SMAD neural induction method was applied, treating ~80% density hiPSCs with neural induction medium (NIM) from day 0 (D0) to day 7, with daily medium changes. Starting from D8, the medium was changed daily to fresh N2 medium containing RA (100 nM), purinamine (PUR, 1 μM) and WIN (1 μM). Starting on D12, cells were manually detached and suspended in ultra-low attachment plates in N2B27 medium containing RA (100 nM), PUR (1 μM) and WIN (1 μM), with medium changes every other day. From D20, the spheroids were further cultured in suspension in PDGF-AA medium. On D30, swollen spher...

Embodiment 2

[0151] Example 2 WIN-derived OPC transplantation improves motor recovery in SCI mice

[0152] To investigate the potential of WIN-derived OPCs as a treatment for SCI, the inventors transplanted OPCs into lesions of SCI mice and assessed motor function using BMS scales, such as Figure 8 A and 8B are shown. After SCI, mice were paralyzed and lost hindlimb mobility (BMS score = 0). On the 7th day after SCI, the OPCs in the WIN group or the control group were radiotherapy to the SCI lesions. The vehicle group received the same volume of saline at the lesion site. After the cell transplantation, the function of the hind limbs of the mice gradually recovered. From the second week after transplantation, the hindlimb motor function of the mice in the WIN group was significantly improved compared with the vehicle group. In the control group, the motor function of the hindlimbs of the mice was significantly improved after the 4th week of cell transplantation, which was significantl...

Embodiment 3

[0154] Example 3 Transplantation of WIN-derived OPCs into SCI mice ameliorated their demyelination

[0155] To demonstrate the myelin sheath at the epicenter of the lesions in each group of mice, the inventors performed LFB staining on coronal sections of the spinal cord. The results showed that the area s of LFB+ was significantly reduced in the vehicle group compared with the sham group, indicating that demyelination occurred after SCI ( Figure 8 E and 8F). Although implantation of OPCs at the lesion site can increase LFB+ area, improve demyelination and maintain tissue integrity. The LFB+ area s of the WIN group was larger than that of the control group, indicating that the WIN-derived OPCs had better remyelination effects compared with the control OPCs ( Figure 8 E and 8F), n = 5, p vs. ctrl-OPC), p vs. Vehicle).

[0156] In order to observe the myelinated axons at the lesion site more microscopically, the inventors observed by transmission electron microscopy (TE...

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Abstract

The invention provides a preparation method and application of oligodendrocytes. The presented protocol recapitulates the major steps of oligodendrocyte differentiation in significantly shorter time than the 75-95 days required for previous protocols. Induction of NSC differentiation by the cannabinoid receptor agonist WIN55212‑2 promotes OLIG2+ OPC induction. WIN-derived OLIG2+ progenitors differentiate into PDGFRα+ OPCs, are highly migratory, and can further differentiate into mature OLs with robust myelinating capacity. When WIN-derived OPCs were transplanted into the lesion site, the locomotor ability of spinal cord injured (SCI) mice was significantly improved from the 2nd week after transplantation. Immunostaining results showed that WIN-derived OPCs could differentiate into mature OLs and myelinate damaged axons. The method and product of the present invention have wide application in the field of cell therapy, especially have remarkable therapeutic effect on spinal cord injury.

Description

technical field [0001] The invention belongs to the field of stem cell therapy, and in particular relates to a preparation method and application of oligodendrocytes. Background technique [0002] Human induced pluripotent stem cells (iPSCs) can be reprogrammed from human skin, blood and other somatic cells in vitro, and have the same infinite proliferation ability as human embryonic stem cells (ESCs), and in vitro The ability to differentiate into almost all functional cells, including neural stem cells (NSCs). This feature of iPSCs successfully circumvents ethical issues and has a wide range of applications in stem cell therapy. [0003] Oligodendrocytes (OLs) are central nervous system cells present in vertebrates. They produce lipid-rich lamellar myelin sheaths that coat neuronal axons and create defined segments of electrical insulation to maximize action potential conduction velocity. Myelin is also important for axonal integrity and survival, and it has been shown ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/079A61K35/30A61P25/00A61P9/10A61P25/28A61P25/16
CPCA61K35/30A61P9/10A61P25/00A61P25/28A61P25/16C12N5/06
Inventor 郭莹高宏
Owner SHENZHEN EXONEUGLIA BIOMEDICAL TECH CO LTD
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