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Preparation method and application of oligodendroglia cells

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

Active Publication Date: 2022-04-01
深圳市夏同生物医药科技有限公司
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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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  • Preparation method and application of oligodendroglia cells
  • Preparation method and application of oligodendroglia cells
  • Preparation method and application of oligodendroglia cells

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) from Guangzhou Institute 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 5% incubator.

[0115]To generate PAX6+ / NESTIN+ / SOX2+ NSCs, a dual-SMAD neural induction method was applied, hiPSCs at ~80% density were treated with neural induction medium (NIM) from day 0 (D0) to day 7, and the medium was changed daily. Starting from D8, the medium was replaced daily with fresh N2 medium containing RA (100 nM), purinamine (PUR, 1 μM) and WIN (1 μM). Starting from 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. Starting from D20, the spheroids were further cultured in suspension in PDGF-AA medium. On D30, ...

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 the lesions of SCI mice and assessed motor function using BMS scales, 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 of the WIN group or the control group were radiotherapyed to the SCI lesion site. The vehicle group received the same volume of saline at the lesion site. After the cell transplantation, the function of the mice's hind limbs gradually recovered. From the second week after transplantation, the motor function of the hind limbs of mice in the WIN group was significantly improved compared with that in 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...

Embodiment 3

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

[0155] To demonstrate myelination at the epicenter of 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, suggesting that demyelination occurred after SCI ( Figure 8 E and 8F). While implanting OPCs into lesion sites 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 WIN-derived OPCs had a better remyelination effect compared with control OPCs ( Figure 8 E and 8F), n = 5, p vs. ctrl-OPC), p vs. Vehicle).

[0156] In order to observe the myelinated axons in the lesion more microscopically, the inventors observed through a transmission electron microscope (TEM). First, the number of ...

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Abstract

The invention provides a preparation method and application of oligodendroglia cells. The provided regimen reproduces the major step of oligodendrocyte differentiation in a time that is significantly shorter than 75-95 days required for previous regimens. The NSC differentiation is induced through a cannabinoid receptor agonist WIN55212-2, and the induction of OLIG2 + OPC is promoted. The WIN-derived OLIG2 + progenitor cells are differentiated into PDGFR alpha + OPCs, have high mobility, and can be further differentiated into mature OL with strong myelination ability. When the WIN-derived OPC is transplanted to a diseased region, the athletic ability of a spinal cord injury (SCI) mouse is significantly improved from the second week after transplantation. Immunostaining results show that the WIN-derived OPC can be differentiated into mature OL, so that damaged axons are myelinated. The method and the product disclosed by the invention are widely applied to the field of cell therapy, and particularly have a remarkable treatment 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 (iPSC) can be reprogrammed from human skin, blood and other somatic cells in vitro, and have similar unlimited proliferation ability to human embryonic stem cells (ESC), and in vitro The ability to differentiate into almost all functional cells, including neural stem cells (NSCs). The characteristic of iPSC has successfully bypassed ethical issues and has a wide range of applications in stem cell therapy. [0003] Oligodendrocytes (OL) are central nervous system cells that exist in vertebrates. They produce a lipid-rich lamellar myelin sheath that coats neuronal axons and creates a defined segment of electrical insulation to maximize action potential conduction velocity. Myelin is also important for axonal integrity and survival, and it has b...

Claims

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Application Information

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IPC IPC(8): C12N5/079A61K35/30A61P25/00A61P9/10A61P25/28A61P25/16
CPCA61K35/30C12N2506/45C12N5/0622C12N2501/385A61P25/28
Inventor 郭莹高宏
Owner 深圳市夏同生物医药科技有限公司
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