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Novel method for inducing pluripotent stem cells to directionally differentiate into in-vitro crystalline lenses

A technology of pluripotent stem cells and directed differentiation, which is applied in the field of in vitro induced lens body preparation, can solve the problems of lens body limitation, unable to transition to human lens, unable to form a good shape with optical functions, etc., and achieves wide application prospects, The effect of good light transmission

Inactive Publication Date: 2016-01-13
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0006] At present, research groups at home and abroad have obtained corpuscles with a preliminary lens structure by placing the lens capsule in the aqueous humor in animal experiments, but due to the limitation of material sources, they cannot be transferred to the human lens.
In the study of human lens development, a research team has realized the directional differentiation of embryonic stem cells into lens epithelial cells and lens fiber cells in vitro. This directional differentiation process partially reproduces the development process of the lens, but its application is due to the inability to form a good shape. Optically functional lens bodies are limited

Method used

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  • Novel method for inducing pluripotent stem cells to directionally differentiate into in-vitro crystalline lenses
  • Novel method for inducing pluripotent stem cells to directionally differentiate into in-vitro crystalline lenses
  • Novel method for inducing pluripotent stem cells to directionally differentiate into in-vitro crystalline lenses

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Embodiment 1

[0054] A novel method for directed differentiation of induced pluripotent stem cells into lens bodies, comprising the following steps:

[0055] (1) Directed induction of pluripotent stem cells into primary neuroectodermal cell clusters

[0056] Coat a six-well plate with matrigel solution and store at 37°C, 5% CO 2 Place it in an incubator with saturated humidity for 50 minutes, then absorb the coating solution, and wash it once with DMEM / F12 medium; then seed the pluripotent stem cells on a six-well plate and culture them with mTesR (human embryonic stem cell medium) cultured in liquid for 5 days to obtain primary neuroectoderm cell mass;

[0057] The matrigel solution was embryonic stem cell matrigel (BD matrigel TM hESC-qualifiedMatrix) is formed by dissolving in DMEM / F12 culture medium, and the volume ratio of embryonic stem cell matrigel is 0.8%; the human growth factor contained in mTesR culture medium is noggin of 90ng / ml;

[0058] Under the microscope, it can be obs...

Embodiment 2

[0068] A novel method for directed differentiation of induced pluripotent stem cells into lens bodies, comprising the following steps:

[0069] (1) Directed induction of pluripotent stem cells into primary neuroectodermal cell clusters

[0070] Coat a six-well plate with matrigel solution and store at 37°C, 5% CO 2 Place it in an incubator with saturated humidity for 1 hour, then absorb the coating solution, and wash it once with DMEM / F12 medium; then inoculate pluripotent stem cells on a six-well plate and culture them with mTesR medium for 6 days to obtain primary Neuroectodermal cell mass;

[0071] The matrigel solution was embryonic stem cell matrigel (BD matrigel TM hESC-qualifiedMatrix) is formed by dissolving in DMEM / F12 culture medium, and the volume ratio of embryonic stem cell matrigel is 1.0%; the human growth factor contained in the mTesR culture medium is noggin of 100ng / ml;

[0072] Under the microscope, it can be observed that the obtained primary ectoderm ce...

Embodiment 3

[0082] A novel method for directed differentiation of induced pluripotent stem cells into lens bodies, comprising the following steps:

[0083] (1) Directed induction of pluripotent stem cells into primary neuroectodermal cell clusters

[0084] Coat a six-well plate with matrigel solution and store at 37°C, 5% CO 2Place it in an incubator with saturated humidity for 10 minutes, then absorb the coating solution, wash it once with DMEM / F12 medium; then inoculate pluripotent stem cells on a six-well plate and culture them with mTesR medium for 7 days to obtain primary neurons. Ectodermal cell mass;

[0085] The matrigel solution was embryonic stem cell matrigel (BD matrigel TM hESC-qualifiedMatrix) is formed by dissolving in DMEM / F12 culture medium, and the volume ratio of embryonic stem cell matrigel is 1.2%; the human growth factor contained in mTesR culture medium is noggin of 110ng / ml;

[0086] Under the microscope, it can be observed that the obtained primary ectoderm cel...

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Abstract

The invention relates to a novel method for inducing pluripotent stem cells to directionally differentiate into in-vitro crystalline lenses and aims to form the in-vitro crystalline lenses which are good in form and have an optical function. According to the technical scheme, the method includes the steps of firstly, directionally inducing the pluripotent stem cells to differentiate into primary neuroderm cell clusters; secondly, separating and selecting the primary neuroderm cell clusters; thirdly, inducing the neuroderm cells to differentiate into primary in-vitro crystalline lenses; fourthly, inducing the primary in-vitro crystalline lenses to differentiate into mature in-vitro crystalline lenses. The in-vitro crystalline lenses obtained by the method are applicable to researches of crystalline lens embryo development mechanisms, researches of congenital cataract pathogenesis and screening of cataract-related medicine.

Description

technical field [0001] The invention relates to the technical field of cell biology, in particular to a preparation method for inducing lens bodies in vitro. Background technique [0002] Pluripotent stem cells (pluripotent stem cells) include embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs). Inhibited mice formed teratomas with inner, middle and outer three germ layers. [0003] Pluripotent stem cells play a very important role in the fields of drug research, disease mechanism research and regenerative transplantation. Scholars at home and abroad have used pluripotent stem cells to induce differentiation into specific cells or tissue precursors as raw materials for drug toxicity screening, which has become the first step in clinical trials. In the field of regenerative medicine, scholars at home and abroad have even successfully used pluripotent stem cell technology combined with in vitro directed differentiation and in vivo injection to achieve rege...

Claims

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Application Information

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IPC IPC(8): C12N5/071A61L27/38
Inventor 姚克傅秋黎秦祯蔚
Owner ZHEJIANG UNIV
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