Method for preparing heart valve endothelial cells by inducing pluripotent stem cell differentiation, and application of heart valve endothelial cells

Abstract

The invention discloses a method for preparing heart valve endothelial cells by inducing differentiation of pluripotent stem cells, and application of the heart valve endothelial cells. The method comprises the following steps: planting pluripotent stem cells in a culture dish coated with matrix glue in a tiled manner, and culturing the pluripotent stem cells by using Essential 8 to enable the pluripotent stem cells to be attached to the wall of the culture dish; and culturing the pluripotent stem cells in a differential medium Essential 6 containing Wnt3a and bone morphogenetic protein 4, culturing the pluripotent stem cells in a differential medium Essential 6 containing epidermal growth factors and bone morphogenetic protein 4, digesting mesoderm cells of the heart, and putting the cardiac mesoderm cells in the culture dish into a differential medium Essential 6 containing bone morphogenetic protein 4, transforming growth factors and endothelial growth factors for continuous cultureto obtain heart valve endothelial cells. The method disclosed by the invention is high in differentiation efficiency, and more than 80% of valvular endothelial cells can be obtained under the condition that flow sorting is not carried out, so that sufficient seed cell sources can be guaranteed.

Description

technical field [0001] The invention relates to the field of stem cell differentiation, in particular to a method for inducing differentiation of pluripotent stem cells to prepare heart valve endothelial cells and its application. Background technique [0002] During a heart beat, blood flows from the low-pressure atria to the high-pressure ventricles, and eventually to the major arteries throughout the body. The heart valves act as one-way valves to maintain the one-way flow of blood. When the heart contracts, the mitral valve, which connects the left atrium to the left ventricle, and the tricuspid valve, which connects the right atrium to the right ventricle, prevent backflow of blood from the ventricles to the atria; when the heart relaxes, the aortic valve connects the left ventricle to the aorta , The pulmonary valve, which connects the right ventricle and the pulmonary artery, prevents the reverse flow of blood from the artery to the ventricle. So as to ensure the no...

AI Technical Summary

Problems solved by technology

[0008] (1) The biological valve is easy to decay and has a short lifespan;

[0009] (2) Calcification is prone to occur, so biological valves often face reoperation due to damage (Fu Jie et al., 2011)

[0011] There are many reasons for the lack of seed cells. The first is that heart valve cells are difficult to separate and cultured in vitro for a long time, so the number of cells is extremely limited, and it is difficult to meet the number requirements for seed cell planting.

The second is that it is difficult to obtain analogs of heart valve endothelial cells because of the lack of previous research on human heart valves.

[0014] (1) The differentiation efficiency is low, and the number of real endothelial cells obtained is very small;

[0015] (2) Most of the previous studies were aimed at obtaining vascular endothelial cells, which are difficult to be used for planting tissue-engineered heart valves due to tissue differences;

Simple differentiation to obtain endothelial cells cannot meet the requirements of heart valve seed cells;

[0017] (4) The real valve mesenchymal cells are derived from valve endothelial cells undergoing endothelial-mesenchymal transition, and these endothelial cells have not undergone endothelial-mesenchymal transition tests, so it is difficult to guarantee that they have this function;

[0018] (5) Valve mesenchymal cells will secrete extracellular matrix. Part of the endothelial cells planted on tissue engineered heart valves should maintain their endothelial properties, and the other part should be transformed into mesenchymal cells and secrete extracellular matrix to strengthen and stabilize The role of tissue engineered flaps, the current differentiated endothelial cells have not been able to do this

[0020] (1) The process of this method is very cumbersome, and the cells obtained in each step need to be sorted by flow cytometry. After the mesoderm cells are obtained, the MESP1-positive cells need to be sorted, and the induced cells need to be sorted by CD31 magnetic beads ;

[0021] (2) The cells after mesoderm sorting need to be plated on mouse embryonic fibroblasts in a fibronectin-coated plate, and then induced, the operation is very complicated and time-consuming;

It shows that the differentiation efficiency is not high, and it will increase the workload at the same time

A large amount of cells are also lost during flow sorting

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