Optimized methods for differentiation of cells into cells with hepatocyte and hepatocyte progenitor phenotypes, cells produced by the methods, and methods for using the cells

• US 10,457,914 B2
• Filed: 09/19/2013
• Issued: 10/29/2019
• Est. Priority Date: 10/31/2008
• Status: Active Grant

• Current Assignee: Katholieke Universiteit Leuven
• Original Assignee: Katholieke Universiteit Leuven
• Inventors: Pauwelyn, Karen, Verfaillie, Catherine M.
• Primary Examiner(s): Ton, Thaian N
• Assistant Examiner(s): Montanari, David A.
• Application Number:
  US14/031,971
• Publication Number:
  US 20140186954A1
• Time in Patent Office: 2,231 Days
• CPC Class Codes: C12N 2501/113 Acidic fibroblast growth factor (aFGF, FGF-1)
• CPC Class Codes: C12N 2501/115 Basic fibroblast growth factor (bFGF, FGF-2)
• CPC Class Codes: C12N 2501/119 Other fibroblast growth factors, e.g. FGF-4, FGF-8, FGF-10
• CPC Class Codes: C12N 2501/12 Hepatocyte growth factor [HGF]
• CPC Class Codes: C12N 2501/155 Bone morphogenic proteins [BMP]; Osteogenins; Osteogenic factor; Bone inducing factor
• CPC Class Codes: C12N 2501/16 Activin; Inhibin; Mullerian inhibiting substance
• CPC Class Codes: C12N 2501/415 Wnt; Frizzeled
• CPC Class Codes: C12N 2506/02 from embryonic cells
• CPC Class Codes: C12N 2506/03 from non-embryonic pluripotent stem cells
• CPC Class Codes: C12N 5/0606 Pluripotent embryonic cells, e.g. embryonic stem cells [ES] embryonic germ cells C12N5/0611, induced pluripotent stem cells C12N5/0696
• CPC Class Codes: C12N 5/067 Hepatocytes
• CPC Class Codes: C12N 5/0696 Artificially induced pluripotent stem cells, e.g. iPS

Abstract

The invention is directed to methods for culturing cells so that the cells are induced to differentiate into cells that express hepatocyte phenotypes and hepatocyte progenitor phenotypes. More particularly, the invention relates to methods for culturing cells so that the cells are induced to differentiate into cells that express a definitive endodermal phenotype, a liver-committed endodermal phenotype, a hepatoblast phenotype, and hepatocyte phenotype. The invention is also directed to cells produced by the methods of the invention. The cells are useful, among other things, for treatment of liver deficiency, liver metabolism studies, and liver toxicity studies.

Claims

1. A cell culture composition, comprising isolated expanded human non-embryonic, non-germ multipotent adult progenitor cells in a culture medium comprising about 5 ng/ml to about 500 ng/ml Wnt3a and about 10 ng/ml to about 1,000 ng/ml Activin A, characterized in that the multipotent adult progenitor cells can differentiate into at least one cell type of each of the endodermal, ectodermal, and mesodermal embryonic lineages, wherein the multipotent adult progenitor cells in the composition are expanded in cell culture for 10-40 doublings prior to being combined with Wnt3a and Activin A, and wherein the amounts of Wnt3a and Activin A are effective to induce the multipotent adult progenitor cells to differentiate into progeny cells with a definitive endoderm phenotype.

2. The composition of claim 1 wherein the multipotent adult progenitor cells are derived from a bone marrow cell preparation.

3. The composition of claim 1 or 2 wherein the multipotent adult progenitor cells express telomerase.

4. The composition of claim 1 or 2 wherein the multipotent adult progenitor cells express oct3/4.

5. The composition of claim 1 or 2 wherein the multipotent adult progenitor cells express oct3/4 and telomerase.