US 2017/0304369 A1 · METHODS OF DIFFERENTIATING STEM CELLS INTO LIVER CELL LINEAGES

Abstract

The present disclosure provides methods and kits for the differentiation of stem cells into relevant liver cell lineages, as well as methods of using the relevant liver 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-β (TGFβ). An additional embodiment provides a method for the differentiation of posterior foregut lineage cells into liver bud progenitors (LB) by one or more activators of TGFβ 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 liver bud progenitors into hepatic progenitors by one or more inhibitors of TGFβ 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.

Claims31

Dependent claims

A method of differentiating cells of the definitive endoderm (DE) lineage into posterior foregut lineage comprising contacting said stem cells with:

one or more retinoic acid activators;
and/or one or more inhibitors of TGFβ signaling, optionally further comprising contacting said stem cells with one or more activators of BMP signaling, optionally further comprising contacting said stem cells with one or more activators of FGF signaling, optionally wherein the cells of the posterior foregut lineage comprise elevated gene expression of posterior foregut lineage markers and decreased expression of dorsal foregut markers relative to undifferentiated cells, optionally wherein the duration of the method is about 1 to 84 hours.

A kit for differentiating

(a) cells of the definitive endoderm (DE) lineage into posterior foregut lineage comprising one or more of the following factors:
one or more retinoic acid activators;
and/or one or more inhibitors of TGFβ signaling;
or
(b) cells of the posterior foregut lineage into liver bud progenitors comprising one or more of the following factors:
one or more activators of TGFβ signaling one or more modulators of Wnt signaling;
and/or one or more activators of cyclic AMP/PKA signaling;
or
(c) liver bud progenitors into hepatic progenitors comprising one or more of the following factors:
one or more inhibitors of TGFβ signaling one or more inhibitors of FGF signaling;
and/or one or more inhibitors of Notch signaling;
or
(d) liver bud progenitors into hepatic progenitors comprising one or more of the following factors:
one or more inhibitors of Notch signaling;
activators of ascorbic acid signaling;
one or more activators of cyclic AMP/PKA signaling;
and/or one or more activators of insulin signaling;
or
(e) hepatic progenitors into perivenous hepatocyte-like cells comprising one or more of the following factors:
one or more inhibitors of Notch signaling;
one or more activators of glucocorticoid signaling;
one or more activators of insulin signaling;
one or more activators of ascorbic acid signaling and/or one or more activators of TGFβ signaling;
or
(f) hepatic progenitors into hepatocytes or hepatocyte-like cells comprising one or more of the following factors:
one or more activators of cyclic AMP/PKA signaling;
one or more activators of glucocorticoid signaling;
one or more activators of insulin signaling one or more activators of ascorbic acid signaling;
and/or one or more activators of Wnt signaling one or more inhibitors of Notch signaling.

26 more elements

A kit for maintaining

(a) liver bud progenitors in a self-renewal state comprising one or more of the following factors:
one or more activators of FGF signaling;
and/or one or more activators of Wnt signaling, optionally further comprising one or more of the following factors;
one or more activators of BMP signaling;
one or more epidermal growth factors;
one or more inhibitors of TGFβ signaling;
and/or one or more inhibitors of Notch signaling;
or
(b) hepatocytes or hepatocyte-like cells in a perivenous state comprising one or more of the following factors:
one or more inhibitors of Notch signaling and/or one or more activators of Wnt signaling, optionally further comprising one or more of the following factors:
one or more inhibitors of TGFb;
and/or one or more activators of estrogen signaling;
one or more inhibitors of cyclic AMP/PKA signaling;
or
(c) hepatocytes or hepatocyte-like cells in a periportal state comprising one or more activators of cyclic AMP/PKA signaling.

12 more elements

Dependencies are inferred from the claim text.

Methods of differentiating stem cells into liver cell lineages

Life of this patent

Methods of differentiating stem cells into liver cell lineages

Life of this patent

Abstract

Claims31