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Marker for evaluating hepatic lineage cell maturity, biomics kit and construction method

A lineage cell and cell maturation technology, applied in the field of markers for evaluating the maturity of hepatic lineage cells, can solve the problems of limited stem cell proliferation ability, low functional hepatocytes, and high labor costs

Active Publication Date: 2021-07-13
SHANGHAI EAST HOSPITAL EAST HOSPITAL TONGJI UNIV SCHOOL OF MEDICINE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Under the two-dimensional culture system, the proliferation ability of stem cells is limited, stem cells are easily lost, and the system has high labor costs, large production losses, and unstable cell batch quality, which is not suitable for the industrial preparation and production of stem cell products.
However, as found in studies of hepatocyte generation from pluripotent stem cells, including embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), the time required for hBTSCs or hHpSCs to mature into hepatocytes takes months, and Often obtain less efficient hepatocytes
At the same time, when culturing biliary tree stem cells, hepatic stem cells and mature hepatocytes in vitro, it is often difficult to predict the developmental state and maturity of hepatic stem cells, and it is impossible to quickly diagnose the cells of mature hepatocytes or liver precursor cells on a large scale. state

Method used

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  • Marker for evaluating hepatic lineage cell maturity, biomics kit and construction method
  • Marker for evaluating hepatic lineage cell maturity, biomics kit and construction method
  • Marker for evaluating hepatic lineage cell maturity, biomics kit and construction method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0157] Example 1. Construction of cell lineage-related "transcription factor-microRNA-target gene" regulatory network

[0158] The method for constructing the regulatory network of "transcription factor-microRNA-target gene" in the process of cell development and maturation lineage mainly includes the following steps ( figure 1 ):

[0159] 1. Obtain cells at different stages of cell lineage; perform microRNA sequencing and RNA-seq sequencing (GSE73114 database in 15 public databases in the aforementioned "1. Preparation of chips and RNA-seq sequencing data of cells at different time series stages in the same lineage" and GSE114974 database after routine processing of sequencing data);

[0160] 2. Through principal component analysis (PCA analysis), it is confirmed that the development of cells does have temporal characteristics;

[0161] 3. Obtain different expression patterns in different cell states through short-time sequence expression analysis (STEM analysis), and obtai...

Embodiment 2

[0176] Example 2. Most of the 17 microRNAs and 23 genes are negatively correlated, and verify the accuracy of the "17microRNA" label

[0177] The inventors analyzed that most of the 17 microRNAs and 23 genes have negative correlations.

[0178] according to image 3 As a result of the PCA analysis in A, it can be seen that the four cell types (biliary tree stem cells (hBTSCs), hepatic stem cells (hHPSCs), hepatic progenitor cells (hHBs) and mature hepatocytes (hAHEPs)) have a very clear timing, so This data is applicable to the "transcription factor-microRNA-target gene" network construction method of the present invention.

[0179] After constructing a network on the public data, the inventors obtained 17 microRNAs and 23 genes, which not only have lineage timing, but also have mutual regulatory relationships. According to the results of 3D_PCA analysis, it can be seen that the differences in timing and maturity of these four cell types can be found only by gene tags or mic...

Embodiment 3

[0182] Example 3. Embryo development-related database verification of the accuracy of the "23 genes" label

[0183] To analyze 23 gene signatures (PIK3R1, PTEN, ACSL1, ANKRD46, CPEB3, CRY2, CSF1R, HAND2, HECW2, MEGF9, NHLRC3, NTF3, PAIP2, PDE4D, PGRMC1, PNRC1, RORA, SLC10A7, SLC8A1, SPRYD4, TIMP3, TMEM135 , TMEM64), the inventors used the following disclosed embryonic development-related databases to obtain cells at different lineage developmental stages, and analyzed the expression of the 23 gene signatures.

[0184] GSE90047: Including cells developed from hepatoblast to hepatocytes during embryonic development, embryonic day 10.5 (E10.5) to embryonic day 18.5 (E18.5);

[0185] GSE132034: including mouse liver organs at various developmental stages, embryo E12.5 → eighth week after birth (W8);

[0186] GSE28892: includes three types of cells: adult hepatic progenitor LPCs (Adult LPCs), differentiated mature hepatocytes (Differentiated Heps), and primary hepatocytes (Primary...

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Abstract

The invention provides a marker for evaluating hepatic lineage cell maturity, a biomics kit and a construction method. According to the invention, a microRNA set and a gene set which have time sequence characteristics and have a regulatory relationship in cell development are elaborated from a new perspective for the first time, a transcription factor-microRNA-target gene regulatory network related to hepatocyte differentiation and maturation regulation is established, and a gene tag and a microRNA tag of hepatolineage specificity are obtained, wherein these tags or combinations thereof can be used as potential new markers for hepatic lineage cells. The invention also provides a biomics kit for the hepatic lineage cells, and a new scheme is provided for large-scale amplification of stable hepatic lineage cells and monitoring of in-vitro culture states of the hepatic lineage cells.

Description

technical field [0001] The present invention relates to the field of biomedicine; more specifically, the present invention relates to a marker for evaluating the maturity of hepatic lineage cells, a dual-omics kit and a construction method. Background technique [0002] Stem cells, as a cell type with self-renewal and multilineage differentiation potential, provide an effective new therapeutic strategy for the treatment of organ function loss caused by end-stage diseases. At present, more than ten kinds of stem cell therapy products have been approved for marketing in the world, but there is no product approved in China. With the gradual improvement of China's stem cell therapy product management standards, 51 stem cell clinical studies in China have been filed with the National Health and Medical Commission, and 3 stem cell drugs have been accepted by the Food and Drug Administration and approved by the IND to enter clinical trials. The liver is the most important organ of...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6888C12N15/113
CPCC12Q1/6888C12Q2600/178C12Q2600/158
Inventor 何志颖刘中民王喜城张文成
Owner SHANGHAI EAST HOSPITAL EAST HOSPITAL TONGJI UNIV SCHOOL OF MEDICINE
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