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Simple, highly-efficient and mechanized inducing method for differentiation of human pluripotent stem cells into retinal tissues

A technology of human pluripotent stem cells and retina, which is applied in the field of programmed induction, can solve the problems of long learning time, poor differentiation homogeneity, and poor experiment repeatability, and achieve the effects of increased acquisition, good structure formation, and improved efficiency

Active Publication Date: 2019-03-05
ZHONGSHAN OPHTHALMIC CENT SUN YAT SEN UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0004] The present invention aims at the limitations of existing induced differentiation schemes, such as not being universally applicable, poor experiment repeatability, poor differentiation homogeneity, long learning time and other shortcomings, and provides a method that can make the induced differentiation and acquisition of retinal tissue an assembly line operation, A simple, efficient and mechanizable induction method for the differentiation of human pluripotent stem cells into retinal tissue that can be produced in a factory for large-scale clinical applications or drug screening

Method used

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  • Simple, highly-efficient and mechanized inducing method for differentiation of human pluripotent stem cells into retinal tissues
  • Simple, highly-efficient and mechanized inducing method for differentiation of human pluripotent stem cells into retinal tissues
  • Simple, highly-efficient and mechanized inducing method for differentiation of human pluripotent stem cells into retinal tissues

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Experimental program
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Effect test

Embodiment 1

[0031] 1. Maintenance culture and passage of human pluripotent stem cells

[0032] ① Cells: SB cell line (hiPSC cells derived from skin fibroblasts; Cellapy, CA4002106)

[0033] ②Reagents and consumables:

[0034] 1) mTeSR1 medium: STEM CELL, #05851, 4°C

[0035] 2) EDTA: Invitrogen, 15575-038, room temperature

[0036] 3) PBS (1X): Gino Biomedical Technology Co., Ltd., 14111202, room temperature

[0037] 4) Matrigel: Corning, 354277, -20°C

[0038] 5) Six-hole plate: FALCON, 353046

[0039] 6) Centrifuge tube: BD FALCON, 352096

[0040] ③Instrument:

[0041] 1) CO 2 Incubator: SANYO, MCO-20A1C

[0042] 2) Inverted microscope: Nikon, TS100

[0043] ④ Step: hPSC cells (SB cell line) are maintained in mTeSR1 medium, and the clones can grow to occupy about 80% of the bottom area of ​​the well in about 4-5 days. Differentiated cells were scraped off under a light microscope before passaging. Aspirate the culture medium and rinse twice with sterile PBS. Add 1ml of 0.5mM...

Embodiment 2

[0104] The most significant technical improvement of the present invention includes two points: first, a single hPSC is used as the starting cell for differentiation, and the number of cells is limited to induce differentiation; second, the expression curve of DKK-1 is increased, and DKK-1 protein is supplemented as needed, To ensure that the differentiation is suitable for most hPSC cell lines.

[0105] 1. Use a single hPSC as the starting cell for differentiation, and limit the number of cells to induce differentiation in order to realize mechanized operation

[0106] The efficiency of induction of differentiation and the number of cell populations with different fates are mainly determined by the size of embryoid bodies and the density after attachment. Therefore, the existing differentiation schemes cannot quantitatively control the size and attachment density of embryoid bodies, which seriously affects the homogeneity of their differentiation and the possibility of large-...

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Abstract

The invention discloses a simple, highly-efficient and mechanized inducing method for differentiation of human pluripotent stem cells into retinal tissues. The inducing method comprises the followingsteps: digesting hPSCs so as to obtain cell pellets, then culturing the cell pellets so as to obtain embryoid bodies, and inducing the embryoid bodies for differentiation so as to obtain 3D retinal tissues. The inducing method is characterized in that the hPSCs are digested to obtain the cell pellets which are cultured to obtain the embryoid bodies, that is, the hPSCs are digested into single cells which are collected to be added into a culture container for cell reaggregation to form the embryoid bodies. The inducing method provided by the invention can enable induced differentiation and acquisition of the retinal tissues to realize pipeline operation, and can realize industrialized production for application in large-scale clinical application or drug screening.

Description

Technical field: [0001] The invention belongs to the technical field of induction and differentiation of stem cells, and the specific technology relates to a program-controlled induction method of differentiation of human pluripotent stem cells to three-dimensional retinal tissue. Background technique: [0002] Human pluripotent stem cells (hPSCs), including embryonic stem cells (Embryonic stem cells, ESCs) and induced pluripotent stem cells (Induced pluripotent stem cells, iPSCs), have the potential for multidirectional differentiation, that is, they can be directed to Differentiate into almost all cells of the body. The breakthrough of stem cell regeneration technology has brought unprecedented opportunities for research on the pathogenesis of refractory diseases, new drug development and cell therapy. Among them, breakthroughs have been made in the key technology of inducing hPSCs to differentiate into retinal cells and even tissues in vitro, and it has become a leader i...

Claims

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Application Information

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IPC IPC(8): C12N5/079
CPCC12N5/0621C12N2501/998C12N2506/02C12N2513/00C12N2506/45
Inventor 葛坚罗子明钟秀风李凯婧
Owner ZHONGSHAN OPHTHALMIC CENT SUN YAT SEN UNIV
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