Human Stem Cell Derived Endothelial Cells, Endothelial-hepatocyte Co-culture System and Uses Thereof

Content Provider	The Lens
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.
Related Links	https://www.lens.org/lens/patent/011-955-058-941-809/frontpage
Language	English
Publisher Date	2019-10-03
Access Restriction	Open
Content Type	Text
Resource Type	Patent
Jurisdiction	United States of America
Date Applied	2017-05-16
Applicant	Agency Science Tech & Res
Application No.	201716302615
Claim	- 29 . (canceled) A method of deriving and maintaining endothelial cells, comprising: (a) culturing lateral plate mesoderm cells under oxygen-deprived condition to obtain endothelial lineage cells; and (b) culturing the endothelial lineage cells from (a) on an extracellular matrix to maintain and expand the endothelial lineage cells; wherein the lateral plate mesoderm cells in (a) comprise splanchnic lateral plate mesoderm cells. The method of claim 30 , wherein the oxygen-deprived condition is carried out under oxygen supply of between 0.5% to 5%, or at about 1%. The method of claim 30 , wherein the lateral plate mesoderm cells are cultured in a first cell culture medium, wherein the first cell culture medium comprises: (a) Fibroblast growth factor (FGF); (b) TGF-β inhibitor; and (c) Vascular endothelial growth factor (VEGF); optionally wherein the lateral plate mesoderm cells are cultured in the first cell culture medium for 3 to 7 days, or for about 5 days. The method of claim 32 , wherein the fibroblast growth factor is fibroblast growth factor 2 (FGF2); optionally wherein the fibroblast growth factor is at a concentration of between 1 to about 10 ng/ml, or at about 4 ng/m The method of claim 32 , wherein the TGF-β inhibitor is 4-[4-(1,3-benzodioxol-5-yl)-5-(2-pyridinyl)-1H-imidazol-2-yl]benzamide (SB 431542); optionally wherein the TGF-β inhibitor is at a concentration of between 1 to 30 μM, or at about 10 μM. The method of claim 32 , wherein the VEGF is VEGF-A; optionally wherein the VEGF-A is of an isoform of VEGF 165 ; optionally wherein the VEGF is at a concentration of between 10 to 100 ng/ml, or at about 50 ng/m The method of claim 32 , wherein the fibroblast growth factor is fibroblast growth factor 2 (FGF2), the TGF-β inhibitor is 4-[4-(1,3-benzodioxol-5-yl)-5-(2-pyridinyl)-1H-imidazol-2-yl]benzamide (SB 431542), and the vascular endothelial growth factor is VEGF 165 ; optionally wherein the FGF2 is at a concentration of between 1 to 10 ng/ml, or at about 4 ng/ml, wherein the SB 431542 is at a concentration of between 1 to 30 μM, or at about 10 μM, and wherein the VEGF 165 is at a concentration of between 10 to 100 ng/ml, or at about 50 ng/ml; optionally wherein the FGF2 is at a concentration of 4 ng/ml, wherein the SB 431542 is at a concentration of 10 μM, and wherein the VEGF 165 is at a concentration of 50 ng/m The method of claim 32 , wherein the first culture medium does not comprise an activator of the Wnt signaling pathway. The method of claim 30 , wherein the endothelial lineage cells to be cultured on the extracellular matrix in (b) are characterized by the expression of one or more markers, wherein the one or more markers comprise at least one marker selected from the group consisting of PECAM-1, CD144 (VE-cadherin), Von Willebrand factor (vWF) and Endothelial NOS (eNOS) and combinations thereof; optionally wherein the endothelial lineage cells to be cultured on the extracellular matrix in (b) are characterized by the expression of PECAM-1, CD144 (VE-cadherin), Von Willebrand factor (vWF) and Endothelial NOS (eNOS). The method of claim 30 , wherein the lateral plate mesoderm cells to be cultured in the extracellular matrix in (b) are further characterized by the uptake of fluorescently-labelled acetylated low-density lipoprotein. A cell co-culture system comprising an endothelial cell culture and a hepatocyte cell culture, wherein the endothelial cell culture is derived using a method of deriving and maintaining endothelial cells, comprising: (a) culturing lateral plate mesoderm cells under oxygen-deprived condition to obtain endothelial lineage cells; and (b) culturing the endothelial lineage cells from (a) on an extracellular matrix to maintain and expand the endothelial lineage cells; wherein the lateral plate mesoderm cells in (a) comprise splanchnic lateral plate mesoderm cells. The cell co-culture system of claim 40 , wherein the hepatocyte cell culture comprises metabolically active hepatocyte cells. The cell co-culture system of claim 40 , wherein the endothelial cell culture and the hepatocyte cell culture are cultured in a co-culture medium, and wherein the co-culture medium comprises a mixture of an endothelial cell culture medium and a hepatocyte cell culture medium in the ratio of 2:1 to 1:2, or at about 1:1. A method of disease modelling or drug testing using a cell co-culture system comprising an endothelial cell culture and a hepatocyte cell culture, wherein the endothelial cell culture is derived using a method of deriving and maintaining endothelial cells, comprising: (a) culturing lateral plate mesoderm cells under oxygen-deprived condition to obtain endothelial lineage cells; and (b) culturing the endothelial lineage cells from (a) on an extracellular matrix to maintain and expand the endothelial lineage cells; The method of claim 43 , wherein the disease modelling comprises modelling of inflammation or drug-induced vascular injury. The method of claim 43 , wherein the drug testing comprises testing the effect of the drug on the endothelial cell culture, testing drug-induced vascular injury, testing hepatotoxicity, predicting vascular protection, predicting drug efficacy or predicting drug safety. The method of claim 45 , wherein the testing of the effect of the drug on the endothelial cell culture drug testing comprises: (a) co-culturing the endothelial cell culture and the hepatocyte cell culture with the drug to obtain hepatocyte metabolized drug; and (b) measuring the effect of the hepatocyte metabolized drug from (a) on the endothelial cell culture. The method of claim 46 , wherein the endothelial cell culture and the hepatocyte cell culture are co-cultured with the drug for 24 hours to 3 days, or for about 48 hours.
CPC Classification	MICROORGANISMS OR ENZYMES;COMPOSITIONS THEREOF;PROPAGATING; PRESERVING; OR MAINTAINING MICROORGANISMS;MUTATION OR GENETIC ENGINEERING;CULTURE MEDIA Investigating Or Analysing Materials By Determining Their Chemical Or Physical Properties
Extended Family	090-371-219-551-198 185-616-485-507-541 011-955-058-941-809 092-144-821-402-411 135-828-156-438-910 182-596-694-908-179
Patent ID	20190300851
Inventor/Author	Cheung Christine Narmada Balakrishnan Chakrapani Goh Yeek Teck
IPC	C12N5/071 G01N33/50
Status	Discontinued
Owner	Agency for Science Technology and Research
Simple Family	090-371-219-551-198 185-616-485-507-541 011-955-058-941-809 092-144-821-402-411 135-828-156-438-910 182-596-694-908-179
CPC (with Group)	C12N5/069 C12N5/067 C12N2501/115 C12N2501/15 C12N2501/165 C12N2502/14 C12N2506/02 C12N2506/45 G01N2500/10 C12N2503/02 G01N33/5005
Issuing Authority	United States Patent and Trademark Office (USPTO)
Kind	Patent Application Publication