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Human stem cell derived endothelial cells, endothelial- hepatocyte co-culture system and uses thereof

An endothelial cell, co-culture technique for use in cell biology and biochemistry that addresses issues such as expensive ethics, slow animal testing, and reduced effectiveness of therapeutic strategies

Inactive Publication Date: 2019-01-04
AGENCY FOR SCI TECH & RES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Thus, the disconnect between in vitro studies, translational animal models, and human clinical studies reduces the effectiveness of the resulting therapeutic strategies
Furthermore, animal testing is a slow and expensive process and raises ethical issues

Method used

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  • Human stem cell derived endothelial cells, endothelial- hepatocyte co-culture system and uses thereof
  • Human stem cell derived endothelial cells, endothelial- hepatocyte co-culture system and uses thereof
  • Human stem cell derived endothelial cells, endothelial- hepatocyte co-culture system and uses thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0117] Example 1: Materials and methods

[0118] hPSC culture and maintenance

[0119] hPSCs were passaged using a mild cell dissociation agent (Stemcell Technologies, Cat. No. 07174) and seeded on Matrigel-coated plates in mTeSR1 medium (Stemcell Technologies, Cat. No. 05850). H9-ESCs and IMR90-iPSCs were purchased from WiCell Institute. BJ-iPSCs were kindly provided by collaborators' laboratories.

[0120] Generation of endothelial cells from hPSCs

[0121] After hPSC colonies attach, they are first induced to drive early mesoderm differentiation for 36 hours using a chemically defined medium supplemented with recombinant human FGF2 (20 ng / ml, R&D Systems (R&D Systems, Cat. No. 233-FB), LY294002 (10 μΜ, Sigma, Cat. No. L9908), and human recombinant BMP4 (10 ng / ml, R&D Systems, Cat. No. 314-BP). Lateral plate mesoderm was further induced for 3.5 days in medium supplemented with human recombinant FGF2 (20 ng / ml) and BMP4 (50 ng / ml), with medium changes every 2 days. O...

Embodiment 2

[0153] Example 2: Derivation of functional endothelial cells from human pluripotent stem cells

[0154] Lateral plate mesoderm is the precursor tissue of the vascular lineage. Lateral plate mesoderm was induced with fibroblast growth factor 2 (FGF2), bone morphogenetic protein 4 (BMP4), and a phosphatidylinositol 3-kinase inhibitor (LY294002) for 5 days ( figure 1 A). A combination of factors is used to drive endothelial specification. Inhibition of transforming growth factor beta (TGF-β) using the small molecule SB431542 enhanced endothelial differentiation of hPSCs, possibly by counteracting the growth of parietal cells that might arise from common cardiovascular progenitors. FGF2 and vascular endothelial growth factor (VEGF) are mitogens for promoting angiogenesis and endothelial development. Hypoxic conditions increase the efficiency of endothelial differentiation because upregulation of hypoxia-inducible factors triggers downstream targets that are important in early v...

Embodiment 3

[0156] Example 3: HPSC-derived endothelial cells respond to inflammatory stimuli

[0157] Inflammation is a hallmark of atherosclerosis. To recapitulate atherosclerosis-associated phenotypes in hPSC-ECs, the inflammatory cytokine interleukin-1β (IL-1β), which is widely implicated in atherosclerosis, was used. After stimulation with recombinant human IL-1β, hPSC-ECs responded in which inflammatory genes were significantly upregulated ( figure 2 A). Nuclear translocation of nuclear factor kappa B (NFκB), which activates a major pro-inflammatory mediator, has been observed in human atherosclerotic lesions. Likewise, nuclear translocation of NFκB was evident in hPSC-ECs following stimulation with IL-1β ( figure 2 B). Interleukin 8 (IL-8) production in the conditioned medium of IL-1β-stimulated hPSC-ECs was significantly higher than that of unstimulated cells ( figure 2 C). In addition to H9-EC, the inventors of the present application also verified that BJ-EC and IMR90-EC...

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Abstract

The present disclosure provides a method of deriving endothelial cells, comprising (a) culturing lateral plate mesoderm cells under oxygen-deprived condition to obtain endothelial lineage cells; and (b) culturing endothelial cells from (a) on an extracellular matrix to maintain and expand the endothelial lineage cells. Also disclosed herein is a cell co-culture system comprising an endothelial cell culture and a hepatocyte cell culture, as well as a microfluidic-based system comprising said cell co-culture system. Also disclosed herein is a method of disease modelling or drug testing using said cell co-culture system or said microfluidic-based system.

Description

[0001] Cross References to Related Applications [0002] This application claims priority to Singapore Provisional Application No. 10201603939U filed on 17 May 2016, the contents of which are hereby incorporated by reference in their entirety for all purposes. technical field [0003] The present invention relates to cell biology and biochemistry, in particular methods of deriving and maintaining cells, especially endothelial cells. The invention also relates to a cell co-cultivation system and its application. Background technique [0004] Cardiovascular disease is a group of conditions of the heart and blood vessels and they include: coronary heart disease (disease of the blood vessels supplying the heart muscle); cerebrovascular disease (disease of the blood vessels supplying the brain); peripheral artery disease (disease of the blood vessels supplying the arms and legs) vascular disease); rheumatic heart disease (damage to the heart muscle and heart valves caused by rhe...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/071G01N33/50
CPCC12N5/067C12N5/069C12N2501/115C12N2501/15C12N2501/165C12N2502/14C12N2506/02C12N2506/45G01N2500/10C12N2503/02G01N33/5005
Inventor 张慧雯B.C.纳马达Y.T.吴
Owner AGENCY FOR SCI TECH & RES
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