Methods of differentiating stem cells into liver cell lineages

Abstract

The invention discloses methods of differentiating stem cells into liver cell lineages. The present disclosure provides methods and kits for the differentiation of stem cells into relevant Iiver cell lineages, as well as methods of using the relevant Iiver cell lineages in screening for a cellular response, a phenotype and in the treatment of a condition. In one embodiment, stem cells are first differentiated into cells of the definitive endoderm lineage, which are differentiated into posterior foregut (PFG) lineage cells by one or more of retinoic acid activators and / or one or more inhibitors of transforming growth factor-[beta] (TGF[beta]). An additional embodiment provides a method for the differentiation of posterior foregut lineage cells into Iiver bud progenitors (LB) by one or more activators of TGF[beta] signalling, and / or one or more modulators of Wnt signalling, and / or one or more activators of cyclic AMP / PKA signaling; and a further embodiment provides a method for the differentiation of Iiver bud progenitors into hepatic progenitors by one or more inhibitors of TGF[beta] signalling and / or fibroblast growth factor (FGF) inhibitors and / or one or more Notch inhibitors. Another embodiment discloses the differentiation of hepatic progenitors into hepatocyte-like cells or perivenous hepatocyte-like cells by one or more of Notch inhibitors and / or activators of glucocorticoid signalling and / or one or more activators of insulin signalling and / or one or more of ascorbic acid signalling activators and / or additional factors. Methods and kits for maintaining LB in self renewal state, hepatocyte-like cells in perivenous or periportal state, as well as surface markers for LB and mid / hindgut (MHG) cells are also disclosed.

Description

[0001] Cross References to Related Applications [0002] This application claims the benefit of priority from Singapore Application No. 10201406436U filed on 8 October 2014, the contents of which are hereby incorporated by reference in their entirety for all purposes. technical field [0003] The present invention generally relates to the field of biotechnology. In particular, the present invention relates to methods for differentiating cells of the definitive endoderm into multiple cell lineages. The invention further relates to kits and media for performing the methods as described herein. Background technique [0004] End-stage liver failure results in severe clinical symptoms including hemorrhage, encephalopathy and eventually death. Notably, the liver primarily contains hepatocytes, which perform a wide variety of functions that are essential to living organisms. These functions include elimination of harmful toxic byproducts from the bloodstream, such as tyrosine me...

AI Technical Summary

Problems solved by technology

[0007] Furthermore, the factors that confer the ability of hepatocytes to engraft, proliferate, and differentiate in vivo remain to be fully examined, among which difficulties in obtaining adult-like hepatocytes are still widely experienced

[0008] In conclusion, although pluripotent stem cells can give rise to hepatocytes with some hepatic function, there remain clear unsurmounted barriers to the progression of these hepatocytes to an adult-like state

Moreover, it remains challenging to generate "true" and transplantable hepatocyte-like cells from these pluripotent stem cells that can engraft and robustly repopulate in the adult liver

Images