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Method for inducing differentiation of human induced pluripotent stem cells into endothelial progenitor cells

A technology of pluripotent stem cells and endothelial progenitor cells, applied in the field of stem cell induced differentiation

Active Publication Date: 2019-11-12
SUN YAT SEN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, there is no method in the prior art that can efficiently induce human induced pluripotent stem cells to differentiate into endothelial cells

Method used

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  • Method for inducing differentiation of human induced pluripotent stem cells into endothelial progenitor cells
  • Method for inducing differentiation of human induced pluripotent stem cells into endothelial progenitor cells
  • Method for inducing differentiation of human induced pluripotent stem cells into endothelial progenitor cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] Example 1 Stage-specific expression of GREM1 during the differentiation and maintenance of hiPSCs into endothelial progenitor cells

[0044] In this example, differentiation was induced according to the following protocol, which included two steps: the first step was to induce undifferentiated hiPSCs to differentiate into mesoderm for 2 days with CHIR99021; the second step was to induce endothelial progenitors with DMEM / F12 medium plus ascorbic acid Cells were grown for 3 days, and then continued to maintain cells ( figure 1 A); clonally grown hiPSCs transformed into endothelial progenitor cells ( figure 1 B) On day 5 of differentiation, the expression of endothelial progenitor cell surface markers CD34, CD31, VEGFR2 and CD144 was detected by immunofluorescence ( figure 1 C); qPCR was used to detect gene expression.

[0045] The experimental results showed that the stem cell markers OCT4, Nanog and SOX2 all decreased after differentiation ( figure 1 D). In contrast,...

Embodiment 2

[0048] Example 2 Knockdown of GREM1 at stage 1 (0-2 days of differentiation) increases the differentiation of hiPSCs into EPCs

[0049] To examine the role of GREM1, GREM1 expression was knocked down using si-GREM1. QPCR results showed that the efficiency of si-GREM1 was above 80% ( image 3 A). WB results confirmed the downregulation of GREM1 at the protein level ( image 3 B, image 3 C).

[0050] When GREM1 was knocked out from day 0 to day 2, FACS results on day 2 showed that CD34 / CD31 increased from (7.21±0.57)% to (10.31±0.53)%, VEGFR2 / CD144 increased from (8.66±0.40) % increases to (11.98±0.75)% ( image 3 D, 3E). Ac-LDL uptake increased in siGREM1 group ( image 3 F, 3G). Tube formation in the siGREM1 group also increased ( image 3 H, 3I).

[0051] Meanwhile, downregulation of GREM1 promoted cell proliferation at this stage. Immunofluorescence of Ki67 expression showed that the positive cells per high-power field of view increased from (37.00±6.97)% to (68....

Embodiment 3

[0052] Example 3 Knockdown of GREM1 in the second stage (2-5 days of differentiation) inhibits the differentiation of hiPSCs into EPCs

[0053] From day 2 to day 5, the expression of GREM1 decreased, and the surface markers of CD34 / CD31 decreased from (19.17±0.52)% to (13.51±0.38)% on day 5, and VEGFR2 / CD144 decreased from (15.60±0.49)% down to (11.33±0.58)% ( Figure 5 A, 5B). The dil-ac-LDL uptake and tube-forming function decreased in the siGREM1 group ( Figure 5 C, 5D, 5E, 5F).

[0054] Simultaneous detection of cell proliferation and apoptosis. Ki67 positive cells decreased from (70.09±1.81)% to (35.00±2.50)% ( Figure 6 A, 6B). The cell cycle showed that the ratio of cells in G1 phase decreased and the ratio of cells in S phase increased in the si-GREM1 group ( Figure 6 C, 6D). PI / AnnexinV results showed that the double positive rate rose from (0.89±0.11)% to (7.58±0.37)% ( Figure 6 E, 6F).

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Abstract

The invention discloses a method for inducing differentiation of human induced pluripotent stem cells into endothelial progenitor cells. The method for inducing differentiation of human induced pluripotent stem cells into endothelial progenitor cells comprises the following steps: 1) maintaining culture of human induced pluripotent stem cells in an mTeSR1 culture medium, carrying out inoculation at a density of 80-90% on a six-well plate coated with matrix adhesive, and adding the mTeSR1 culture broth into Y27632; 2) after 24 hours of inoculation, starting induction of differentiation with a DMEM / F12 medium added into the culture broth at the beginning of the induction of differentiation; and 3) sucking the medium on day 2 of differentiation, adding DMEM / F12 medium, changing DMEM / F12medium every day so as to allow maintenance of cells, adding recombinant protein GREM1 on days 0-2, days 3-5 or days 5-8 of differentiation, and then, detecting cell surface markers so as to obtain CD34+ and CD31+ endothelial progenitor cells. Being adopted, the method for inducing differentiation of human induced pluripotent stem cells into endothelial progenitor cells disclosed by the inventionis capable of efficiently obtaining double-positive (namely CD34+ and CD31+) endothelial progenitor cells; and the differentiation efficiency is up to 22.4-34.7%.

Description

technical field [0001] The invention relates to the technical field of stem cell induction and differentiation, in particular to a method for inducing human induced pluripotent stem cells to differentiate into CD34+ and CD31+ endothelial progenitor cells. Background technique [0002] The current method of inducing the differentiation of stem cells into cardiovascular lineage cells in vitro mainly uses the mechanism of embryonic development to simulate the process of embryonic heart development. Cardiac and vascular tissue can be identified during the early stages of vertebrate embryonic development. When the embryo sac of the three germ layers is formed, the mesoderm progenitor cells, cardiac progenitor cells and vascular progenitor cells are activated by different induction signals to start the development of the heart and blood vessels 【1】 . Cardiogenesis and angiogenesis in mesoderm are promoted by highly conserved molecular mechanisms such as intercellular signaling a...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/071
CPCC12N5/069C12N2506/45
Inventor 向秋玲陈海璇张圳张弛
Owner SUN YAT SEN UNIV
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