Method for promoting long-term stable passage of pancreas islet precursor cells

A technology of precursor cells and islet cells, applied in the biological field, can solve the problems of unclear molecular mechanism and difficulty in obtaining the culture system of islet precursor cells, and achieve high uniformity, improved purity, and promoted expression

Active Publication Date: 2022-04-12
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, since the molecular mechanism of self-renewal of human islet precursor cells is still unclear, the in vitro culture and expansion of...

Method used

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  • Method for promoting long-term stable passage of pancreas islet precursor cells
  • Method for promoting long-term stable passage of pancreas islet precursor cells
  • Method for promoting long-term stable passage of pancreas islet precursor cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0061] Example 1 Differentiation of human pluripotent stem cells into islet precursor cells

[0062] First, we use human pluripotent stem cell medium to culture human pluripotent stem cells. The culture medium for human pluripotent stem cells contains 88% DMEM, 10% KSR, 1% NEAA, 1% mixed solution of penicillin and streptomycin, 0.055mM β-mercaptoethanol, and 10ng / mL bFGF. The human pluripotent stem cell lines used include MEL1 human embryonic stem cell line, H9 human embryonic stem cell line and human induced pluripotent stem cell line (hiPSC).

[0063] Afterwards, human pluripotent stem cells were differentiated into islet precursor cells using 2D or 3D differentiation methods. A 3D differentiation method for obtaining islet precursor cells by differentiating human pluripotent stem cells, the steps are as follows:

[0064] 1) Cultivate human pluripotent stem cells in a 10 cm culture dish for four days, use human pluripotent stem cell medium, and change the medium every day;...

Embodiment 2

[0075] Example 2 Expansion of human islet precursor cells by basal medium

[0076] Try expanding islet precursors using defined media. We applied real-time quantitative polymerase chain reaction (RT-qPCR) and immunofluorescence staining to identify the pancreatic differentiation process ( figure 1 A-C). Differentiated to the islet precursor stage, the results of flow cytometry fluorescence analysis (FACS) showed that the percentage of PDX1 and NKX6.1 double positive cells was about 50-60% ( figure 1 D). We then attempted to expand these human islet precursors with the aim of generating large numbers of cells suitable for downstream assays and applications. We chose the basal medium containing 97% DMEM, 1% penicillin-streptomycin mixed solution, 1X B27, 50ng / mL EGF, 10ng / mLbFGF, 10μM 616452. However, under the culture of basal medium, the percentage of PDX1 and NKX6.1 double positive cells decreased significantly from about 60% to less than 20% after 3-5 passages ( figure ...

Embodiment 3

[0077] Example 3 Screening of small molecules that promote the expansion of human islet precursor cells

[0078] We performed a chemical small molecule screen to identify small molecules that can help maintain efficient expansion of PDX1 and NKX6.1 double positive human islet precursors ( figure 2 A). First, we used basal medium (EF6 medium) to subculture the human islet precursor cells obtained from directed differentiation for 2-3 passages to generate a sufficient number of cells for the next step of chemical small molecule screening. Subsequently, we seeded the cells into 24-well plates, and then treated them with small molecules from the chemical small molecule library, changing the medium every 3 days. The chemical small molecule library mainly contains biologically active small molecule inhibitors targeting epigenetic and signaling pathways ( figure 2 A, B). After 7 days of treatment, we performed immunofluorescence staining on the cells to identify PDX1 and NKX6.1 ...

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Abstract

The invention relates to a method for promoting long-term stable passage of pancreas islet precursor cells and preparing pancreas islet cells and pancreas islet organs with mature functions in a large scale. According to the method, a culture medium at least containing a BET inhibitor and a TGF beta inhibitor is adopted to culture the pancreas islet precursor cells, so that the pancreas islet precursor cells can keep original characteristics after multiple passage, and the obtained pancreas islet cells and pancreas islet organs also have normal functions. Therefore, the islet precursor cells with stable and uniform quality can be massively prepared by the method disclosed by the invention, enough islet cells and islet organs can be further prepared, and the islet precursor cells can be used for disease modeling, drug screening and diabetes treatment and have a very wide application prospect.

Description

technical field [0001] The application relates to a method for promoting long-term and stable subculture of islet precursor cells, which belongs to the field of biotechnology. Background technique [0002] Diabetes is a chronic disease with high incidence in the world, which seriously affects the health of hundreds of millions of people and consumes a lot of medical resources. Islet transplantation holds great promise for the treatment of diabetes, but faces many constraints, notably the shortage of organ donor supplies and associated post-transplantation immunosuppression. Human pluripotent stem cells (hPSCs), including human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs), have the potential to differentiate into all cell types in the human body. In the past 20 years, stem cell differentiation technology has made great progress. Human pluripotent stem cells can be gradually differentiated into definitive endoderm cells (definitive endoderm, ...

Claims

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Application Information

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IPC IPC(8): C12N5/071C12N5/0735C12N5/074C12Q1/02A61K35/39A61P3/10
CPCC12Q1/02A61P3/10C12N5/06A61K35/39
Inventor 祝赛勇马晓洁
Owner ZHEJIANG UNIV
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