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Preparation method and application of pancreatic progenitor cells

A technology of pancreatic progenitor cells and cells, which is applied in the field of preparation of pancreatic progenitor cells, can solve problems such as the inability to elucidate the complexity of the molecular network of pancreatic organs

Pending Publication Date: 2021-08-10
UNIVERSITY OF MACAU
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0004] However, the complexity of the molecular network in pancreatic organogenesis has not yet been elucidated, and deciphering the molecular mechanisms of pancreatic differentiation will facilitate the generation of protocols for the in vitro differentiation of hPSCs to mature, functional pancreatic progenitors and pancreatic β cells

Method used

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  • Preparation method and application of pancreatic progenitor cells
  • Preparation method and application of pancreatic progenitor cells
  • Preparation method and application of pancreatic progenitor cells

Examples

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preparation example Construction

[0021] First, an embodiment of the present invention provides a method for preparing pancreatic progenitor cells, which includes: using a differentiation inducer to induce the differentiation of hindgut cells into pancreatic progenitor cells;

[0022] Such differentiation-inducing agents include casein kinase 1 inhibitors.

[0023] The "posterior hindgut cell" in this article is the same as the posterior foregut cell.

[0024] Casein kinase 1 inhibitors, referred to as CK1 inhibitors for short, the inventors have found through research that CK1 inhibitors play a key role in the process of inducing posterior segment cells to differentiate into pancreatic progenitor cells. In the case of no serum addition (that is, the chemical composition is determined), it can effectively promote the differentiation of posterior segment cells into pancreatic progenitor cells.

[0025] In some embodiments, "using a differentiation-inducing agent to induce the differentiation of the hindgut cel...

Embodiment 1

[0087] To verify the effect of different CK1 inhibitors in the preparation of pancreatic progenitor cells.

[0088] In this example, four CK1 inhibitors, D4476, LH 846, PF 4800567 and PF 5006739, were selected and treated on the 11th to 13th day of differentiation to detect their effects on the differentiation of pancreatic progenitor cells and induce human embryonic stem cells to differentiate into pancreatic The method for progenitor cells is as follows:

[0089] Human embryonic stem cells H1 were cultured in E8 medium, and fresh medium was changed every day, and subcultured when the cell density reached 70%-80%; first, wash twice with DPBS-EDTA, and incubate at room temperature for the third time for 5 minutes, and absorb DPBS -EDTA, add E8 medium containing 5 μM ROCK inhibitor, resuspend the cells, spread them in a 24-well plate pre-coated with Matrigel at a density of 1:3-1:6; change to E8 after the cells adhere to the wall for 1 day The medium continues to be cultured; ...

Embodiment 2

[0100] The embodiment of the present invention provides a method for preparing pancreatic progenitor cells, which includes the following steps:

[0101] Human embryonic stem cells H1 were cultured in E8 medium, and fresh medium was changed every day, and subcultured when the cell density reached 70%-80%; first, wash twice with DPBS-EDTA, and incubate at room temperature for the third time for 5 minutes, and absorb DPBS -EDTA, add E8 medium containing 5 μM ROCK inhibitor, resuspend the cells, and spread them into 24-well plates pre-coated with Matrigel at a density of 1:3-1:6. One day after the cells adhered to the wall, the E8 medium was changed to continue culturing. When the cells grew to 40%-50%, they began to differentiate. On the first day of differentiation, E5 differentiation medium containing CHIR99021 with a final concentration of 5 μM and Activin A with a final concentration of 100 ng / ml was added (E8 medium removed TGFβ, FGF2 and insulin , and add Chemically Define...

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Abstract

The invention discloses a preparation method and application of pancreatic progenitor cells, and relates to the technical field of stem cell differentiation regulation, and the preparation method comprises the following steps: inducing posterior segment cells of a posterior intestine to differentiate into pancreatic progenitor cells by adopting a differentiation inducer; the differentiation inducer comprising a casein kinase 1 inhibitor. The inventor finds that the casein kinase 1 inhibitor plays an important role in the differentiation process of pancreatic progenitor cells, and can promote the acquisition of the pancreatic progenitor cells and / or pancreatic beta cells in a serum-free induction environment, thereby providing a new way for effectively acquiring the pancreatic progenitor cells and the pancreatic beta cells. Meanwhile, a foundation is laid for treating diabetes mellitus or other pancreatic diseases by a pancreatic cell replacement therapy.

Description

technical field [0001] The invention relates to the technical field of stem cell differentiation regulation, in particular to a preparation method and application of pancreatic progenitor cells. Background technique [0002] Pluripotent stem cells (hPSCs) are a type of pluripotent cells with self-renewal and self-replication capabilities. Under certain conditions, it can differentiate into a variety of APSC pluripotent cells. Pluripotent stem cells (HSC) have the potential to differentiate into a variety of cell tissues, but they have lost the ability to develop into a complete individual, and their developmental potential is limited. [0003] Pancreatic progenitor cells are the precursors of insulin-secreting β cells, which can be derived from human pluripotent stem cells (hPSCs) in vitro, which solves the problem of cell resource shortage in the process of cell replacement therapy for diabetes. [0004] However, it has not been possible to elucidate the complexity of the ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/071
CPCC12N5/0678C12N2501/727
Inventor 陈国凯张宇萌
Owner UNIVERSITY OF MACAU
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