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Culture solution for inducing multifunctional stem cells to differentiate into dopamine progenitor cells and differentiation method

A multifunctional stem cell and dopaminergic technology, applied in the field of stem cell therapy, can solve the problems of poor differentiation efficiency, high cost, and low purity

Active Publication Date: 2021-04-30
SHANGHAI AISAER BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In the former approach, induced pluripotent stem cells are treated with CK1 inhibitors and bone morphogenetic protein inhibitors, neural stem cells are identified by measuring neural stem cell markers of treated induced pluripotent stem cells, and then at least one dopaminergic progenitor cell-inducing compound Manipulating neural stem cells and analyzing final differentiated cells for dopaminergic progenitor markers; this method is time-consuming, complicated, and inefficient
The latter method is used in the reported literature and patents to perform directed differentiation by adding a specific cytokine mixture in a precise time sequence during the differentiation process. These differentiation methods are costly, take a long time and are complicated.
Therefore, for the shortcomings of the existing technology: there are uncertain culture factors (such as serum), long induction period, complicated process and uncertainty, poor differentiation efficiency, low purity, etc.

Method used

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  • Culture solution for inducing multifunctional stem cells to differentiate into dopamine progenitor cells and differentiation method
  • Culture solution for inducing multifunctional stem cells to differentiate into dopamine progenitor cells and differentiation method
  • Culture solution for inducing multifunctional stem cells to differentiate into dopamine progenitor cells and differentiation method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1 and comparative example 1

[0029] The preparation of embodiment 1 and comparative example 1 dopaminergic progenitor cell culture fluid

[0030] The culture medium is a basal medium mixed with DMEM / F12 medium and Neurobasal medium, and additional components added to the basal medium. Add components and their contents as shown in Table 1:

[0031] Table 1 Components and content of dopaminergic progenitor cell culture medium

[0032]

[0033]

[0034] Remarks: All substances mentioned in Table 1 are purchased from the market

[0035] In Table 1, 50× and 100× refer to the dilution of the purchased stock solution by 50 times and 100 times, and 1× refers to the liquid diluted 100 times.

Embodiment 2

[0036] Example 2 Inducing pluripotent stem cells to differentiate into dopaminergic progenitor cells

[0037] 1. Coating of cell culture plate

[0038] Dilute laminin-521 and laminin-111 with phosphate buffered saline (DPBS) containing calcium and magnesium ions to a concentration of 14 μg / mL, mix them at a ratio of 1:2 by volume, and mix them at 2 μg / cm 2 Concentrations were spread on cell culture plates at 37°C, 5% CO 2 Conditional coating was performed for 2 hours to obtain coated cell culture plates.

[0039] 2. Differentiation and culture of induced pluripotent stem cells

[0040] Cells were fused to 80% density of induced pluripotent stem cells, washed with DPBS at a volume of 2 mL / well, discarded DPBS, added 1 mL / well of Accutase digestion solution, and placed at 37°C for 5 minutes. After the cells shrank into balls or partially de-adhered to the wall, add 2 times the volume of the culture solution of Example 1 (the culture solution of Comparative Example 1 was also ...

Embodiment 3

[0045] Example 3 Small molecule compounds increase the production of dopaminergic progenitor cells

[0046] Carry out the directed differentiation of dopaminergic progenitor cells by the differentiation culture method of Example 2, respectively use the culture solution of Comparative Example 1 and Example 1, collect the cells in 1 well of a 12-well plate, count by a cell counter, and compare the obtained dopamine number of progenitor cells. The result is as image 3 shown.

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Abstract

The invention discloses a culture solution for inducing multifunctional stem cells to differentiate into dopamine progenitor cells and a differentiation method. The culture solution comprises a DMEM / F12 culture medium and a Neurobaal culture medium in a volume ratio of 1: (0.9-1.3), and an N2 supplement, a B27 supplement, Lascorbic acid, Lglutamine, a GSK3 beta inhibitor, a TGF beta inhibitor, a BMP inhibitor, a fibroblast growth factor, a human leukemia inhibition factor hLIF, heparin Heparin and a nutritional factor required for growth of dopaminergic progenitor cells. The culture solution disclosed by the invention can be used for obtaining dopamine progenitor cells by taking induced multifunctional stem cells as raw materials through directional differentiation culture, can be used for direct transplantation or final differentiation into dopamine neurons, and can be used for clinical cell substitution transplantation treatment of PD, in-vitro disease modeling research, drug screening and other biomedical applications. The method has the advantages of being convenient to operate, short in culture time, high in yield, high in differentiation purity, free of animal-derived components in the whole link and the like.

Description

technical field [0001] The invention belongs to stem cell differentiation technology in the technical field of stem cell therapy, and in particular relates to a culture medium and a differentiation method for inducing multifunctional stem cells to differentiate into dopaminergic progenitor cells. Background technique [0002] Parkinson's disease (PD) is a progressive neurodegenerative disease. Although it was discovered 200 years ago, its etiology and underlying cellular and molecular pathogenic mechanisms are still unclear. It is agreed that the motor symptoms of PD are mainly caused by the degeneration and necrosis of dopaminergic neurons in the substantia nigra of the midbrain. Mammalian nervous system generally cannot regenerate after injury, and the therapeutic effect of traditional treatment methods on nervous system injury is extremely limited, so dopaminergic neurons have become an ideal target for cell replacement therapy. [0003] Material from existing patients w...

Claims

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Application Information

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IPC IPC(8): C12N5/0797C12N5/10C12N5/0793
CPCC12N5/0618C12N5/0619C12N2506/45C12N2510/00C12N2500/38C12N2500/32C12N2501/727C12N2501/15C12N2501/155C12N2501/119C12N2501/235C12N2501/91C12N2501/13C12N2501/998C12N2500/44C12N2501/01C12N2501/40C12N2533/52C12N2506/08
Inventor 高歌
Owner SHANGHAI AISAER BIOTECH CO LTD
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