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Methods of generating hepatic macrophages and uses thereof

A technology for macrophages and hepatocytes, applied in the field of deriving and maintaining hepatic macrophages, which can solve the problems of human Kupffer cells source limitation and inability to expand

Pending Publication Date: 2019-11-08
AGENCY FOR SCI TECH & RES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, sources of human Kupffer cells are limited due to donor availability, cost, and cumbersome isolation procedures
Furthermore, primary human Kupffer cells (PHKC) cannot be maintained for extended periods of time or expanded in culture to obtain larger cell numbers

Method used

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  • Methods of generating hepatic macrophages and uses thereof
  • Methods of generating hepatic macrophages and uses thereof
  • Methods of generating hepatic macrophages and uses thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0091] Example 1--Optimization of culture conditions for the differentiation of hPSC-KC

[0092] hPSC-derived monocytes (hPSC-Mon) were differentiated from iPSC-IMR90 according to the method described by Wilgenburg et al. Preliminary results showed that embryoid bodies (EBs) could be formed and maintained in culture under serum-free and feeder-free conditions ( figure 1 A). EBs adhere within 2 weeks, and monocytes can be generated in about 18 days of culture. These monocytes can be harvested weekly from the supernatant of the differentiation culture.

[0093] hPSC-Mon were harvested from the supernatant and differentiated into hepatic macrophages (hPSC-KC) ( figure 1 A). hPSC-Mac used as a control to analyze non-hepatic macrophages Similarities and differences between hepatic macrophages (KC). The results showed that hPSC-Mon could differentiate into adherent hPSC-KC ( figure 1 A).

[0094] To differentiate hPSC-Mon into hPSC-KC, hPSC-Mon were treated with medium cont...

Embodiment 2

[0096] Example 2--marker expression of hPSC-KC

[0097] After generating hPSC-KCs, cells were analyzed for marker expression by gene expression and immunostaining. F4 / 80 has been documented as a representative marker for mouse Kupffer cells but not for human cells. More recently, the combination of CD14 and the classification of CD32, CD68 and CD11 subsets of Kupffer cells has been used to define Kupffer cells in humans. Furthermore, CD163 has been used as a marker of activated macrophages. Therefore, the expression of these markers in hPSC-Mac and hPSC-KC was examined by gene expression studies. The results showed that hPSC-KC expressed CD14, CD163 and CD32 at levels comparable to PHKC ( figure 2 A). CD68 and CD11 expression in hPSC-KC was about 30% of that in PHKC. Marker expression was confirmed by immunostaining ( figure 2 B).

Embodiment 3-

[0098] Example 3--The cytokines produced by hPSC-KC after activation are similar to PHKC but with different

[0099] To activate hPSC-KCs to examine cytokine production, lipopolysaccharide (LPS) was added to the medium during the last 16 hours of culture. Media were collected at the end of the incubation period and analyzed for morphological changes and cytokine production upon LPS activation. The results showed that LPS activation in and hPSC-KC induced typical morphological changes from round to flattened and spread cells ( image 3 A). Importantly, the fold induction in hPSC-KC was in the same range as that of PHKC (25-fold) ( image 3 B). IL-6 production in primary human hepatocytes (PHH) was below detectable levels. The fold increase in TNF4α production in hPSC-KC (33-fold) was similar to that in PHKC (35-fold) ( image 3 C). TNF4α production in PHH following LPS activation was below detectable levels. Compared with PHKC and hPSC-KC, produced much higher leve...

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Abstract

The present disclosure provides a method of deriving hepatic macrophages from stem cell-derived monocytes, through the use of hepatic macrophage culture medium comprising a hepatocyte conditioned medium and a basal medium, wherein the conditioned medium is obtained through culturing hepatocytes in a serum-free culture medium in the presence of an extracellular matrix. Also disclosed is a kit usedfor such a method and hepatic macrophages derived using the method and uses thereof.

Description

[0001] Cross References to Related Applications [0002] This application claims the benefit of priority from Singapore Patent Application No. 10201700068V filed on 5 January 2017, the contents of which are hereby incorporated by reference in their entirety for all purposes. [0003] field of invention [0004] The present invention relates to cell biology and biochemistry, particularly methods of deriving and maintaining hepatic macrophages. Background technique [0005] The liver is responsible for several functions, the most important of which are metabolism, detoxification and protein synthesis. Many of these functions are performed in hepatocytes, the parenchymal cells that make up 60% of all liver cells. However, the liver also contains a large proportion of nonparenchymal cells, including hepatic sinusoidal endothelial cells, hepatic stellate cells, cholangiocytes, and Kupffer cells (KC). Kupffer cells are resident macrophages in the liver. Located in the hepatic si...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0786
CPCC12N5/0645C12N2502/14C12N2506/115C12N2533/90C12N2500/25C12N2500/32C12N2500/36C12N2500/60C12N2506/45
Inventor F·塔斯尼姆余严军
Owner AGENCY FOR SCI TECH & RES
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