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Method for preparing functional bile duct cells by using entoderm stem cells on scale and application of functional bile duct cells

A technology for the differentiation of cholangiocytes and stem cells, applied in biochemical equipment and methods, cell culture active agents, applications, etc., can solve problems that limit the large-scale preparation of mature cholangiocytes, tumor formation, and functional gaps

Pending Publication Date: 2020-06-19
CENT FOR EXCELLENCE IN MOLECULAR CELL SCI CHINESE ACAD OF SCI
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Problems solved by technology

[0004] However, the current protocols for preparing mature cholangiocytes are highly dependent on the extracellular matrix Matrigel, which greatly limits the large-scale preparation of mature cholangiocytes
[0005] Moreover, there are the following problems in the differentiation of pluripotent stem cells: 1) safety issues - any undifferentiated pluripotent stem cells remaining in the differentiation system may form tumors in vivo (teratoma / Teratoma); 2) differentiated cells Functional problems——It is very difficult to directly differentiate into functional cells from pluripotent stem cells. Compared with adult cells, the terminally differentiated cells directly differentiated from pluripotent stem cells have a large gap in function; 3) differentiated cells The problem of purity of pluripotent stem cells: the differentiation and pluripotency of pluripotent stem cells determines that the target cells induced to differentiate will be mixed with a considerable proportion of cell groups of the same and other germ layers, so it is very difficult to purify the target cells. It is difficult to study the interaction between
In addition, current research has found that although embryonic stem cells or induced pluripotent stem cells can be induced to differentiate in vitro to form cholangiocytes with certain functions, their differentiation efficiency is low and cannot be produced on a large scale. At the same time, there is a risk of tumorigenesis

Method used

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  • Method for preparing functional bile duct cells by using entoderm stem cells on scale and application of functional bile duct cells
  • Method for preparing functional bile duct cells by using entoderm stem cells on scale and application of functional bile duct cells
  • Method for preparing functional bile duct cells by using entoderm stem cells on scale and application of functional bile duct cells

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Experimental program
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preparation example Construction

[0162] 1. Preparation and maintenance of endoderm stem cells (EnSCs)

[0163] Human pluripotent stem cells form committed endoderm in vitro induced by high concentrations of Activin A (D’Amour et al., 2006; Nostro et al., 2011). Then, the CXCR4+CD117+ cell population in the committed endoderm was separated by flow cytometry technology, and the endoderm stem cell line was maintained under serum-free culture conditions containing BMP4, bFGF, EGF, and VEGF.

[0164] 2. Large-scale preparation of cholangiocytes from endoderm stem cells (EnSCs)

[0165] The system utilizes endoderm stem cells derived from H9, Hes2, WT-B5-G7 human pluripotent stem cells to prepare cholangiocytes. Under the induction of SFD-based medium, adding bFGF, VEGF, EGF, and BMP4 factors, it completes liver specialization through continuous culture for six days, and forms Hepatic Endoderm (hepatic endoderm) cells. Subsequently, hepatoblasts were formed through six days of directed differentiation under the c...

Embodiment 1

[0186] Example 1 Large-scale preparation of cholangiocytes from endoderm stem cells

[0187] Early studies have shown that in vitro differentiation of human pluripotent stem cells can form cholangiocytes. However, these systems are mostly based on a two-dimensional adherent differentiation system or a differentiation system highly dependent on Matrigel, which limits the large-scale acquisition of cholangiocytes. Here, we used endoderm stem cells as seed cells to prepare cholangiocytes (E-chos) on a large scale in a stirred flask bioreactor ( figure 1 , A). At the same time, after repeated attempts, we invented a differentiation method suitable for endoderm stem cells to develop toward the direction of the bile duct ( figure 1 , B). The results of real-time quantitative PCR showed that endoderm stem cells completed liver specification on the sixth day of directed differentiation, accompanied by the significant down-regulation of endoderm transcription factor SOX17 and EOMES ge...

Embodiment 2

[0188] Example 2 In vitro functional evaluation of E-chos

[0189] Cholangiocytes are epithelial cells in the bile ducts and an important component of liver cells. It can be activated under endogenous or exogenous stimuli. It not only has the ability to synthesize a certain amount of bile, but also regulates the volume and composition of bile through specific transporters. First, we confirmed that the resulting E-chos have bile acid transport activity mediated by polar bile and bile salt receptor (ASBT). Compared with the control group fluorescein FITC, E-chos was able to exclude the absorbed bile fluorescein (CLF) from the cells ( figure 2 , A, B, C). After 10 days of three-dimensional Matrigel culture, E-chos can form a cystic structure, further confirming its bile duct characteristics. Immunofluorescent staining of the tight junction molecule ZO1 and the bile duct functional membrane protein CFTR further proved that the resulting cystic structure had apical-basal polari...

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Abstract

The invention provides a method for preparing functional bile duct cells by using entoderm stem cells on scale and application of the functional bile duct cells, and specifically, the invention provides a method for inducing human entoderm stem cells to be differentiated into bile duct cells and / or bile duct cell populations. The method comprises the following step: culturing entoderm stem cells in a culture system, so as to obtain functional bile duct cells and / or bile duct cell populations. The functional bile duct cells and / or bile duct cell populations prepared by using the method have very high differentiation rates and purity, and in addition, the bile duct cells and / or bile duct cell populations prepared by using the method can be used as in-vitro models of medicine screening and medicine toxicity evaluation.

Description

technical field [0001] The invention relates to the fields of biotechnology and cell therapy. Specifically, the present invention relates to a method for large-scale preparation of functional cholangiocytes using endoderm stem cells and its application. Background technique [0002] The liver is composed of a variety of cell types, including hepatocytes, cholangiocytes, endothelial cells, mesenchymal cells, Kupi cells, and circulating blood cells. Liver parenchymal cells are the main cell components of the liver. They are epithelial-like cells and are mainly involved in detoxification, metabolic regulation and protein synthesis. In addition, another type of epithelial-like cells in the liver are cholangiocytes, which are mainly involved in the formation of intrahepatic bile ducts and extrahepatic bile ducts, and are responsible for the transportation, modification and secretion of bile generated by hepatic cells into the small intestine. [0003] Currently, bile duct disea...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/071C12Q1/02
CPCC12N5/0602C12N5/067G01N33/5044G01N33/5014C12N2501/115C12N2501/165C12N2501/11C12N2501/155C12N2501/237C12N2501/119C12N2501/15C12N2501/12C12N2501/06C12N2501/727C12N2500/38C12N2503/02C12N2503/00G01N2500/10
Inventor 程新冯思思吴佳颖周莹
Owner CENT FOR EXCELLENCE IN MOLECULAR CELL SCI CHINESE ACAD OF SCI
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