Self-renewing endodermal progenitor lines generated from human pluripotent stem cells and methods of use thereof

• US 9,476,030 B2
• Filed: 12/23/2013
• Issued: 10/25/2016
• Est. Priority Date: 06/23/2011
• Status: Active Grant

• Current Assignee: Children's Hospital of Philadelphia (Children's Hospital of Philadelphia Foundation)
• Original Assignee: Children's Hospital of Philadelphia (Children's Hospital of Philadelphia Foundation)
• Inventors: Gadue, Paul, French, Deborah, Cheng, Xin
• Primary Examiner(s): Long, Scott
• Application Number:
  US14/138,963
• Publication Number:
  US 20140141509A1
• Time in Patent Office: 1,037 Days
• US Class Current: 1/1
• CPC Class Codes: C12N 2501/115 Basic fibroblast growth factor (bFGF, FGF-2)
• 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 2501/165 Vascular endothelial growth factor [VEGF]
• CPC Class Codes: C12N 2502/13 connective tissue cells; generic mesenchyme cells, e.g. so-called "embryonic fibroblasts"
• CPC Class Codes: C12N 2506/02 from embryonic cells
• CPC Class Codes: C12N 2506/45 from artificially induced pluripotent stem cells
• CPC Class Codes: C12N 2533/90 Substrates of biological origin, e.g. extracellular matrix, decellularised tissue
• CPC Class Codes: C12N 5/0672 Stem cells; Progenitor cells; Precursor cells; Oval cells
• CPC Class Codes: C12N 5/0678 Stem cells; Progenitor cells; Precursor cells
• CPC Class Codes: C12N 5/068 Stem cells; Progenitors

Abstract

Self-renewing endodermal progenitor lines generated from human pluripotent stem cells and methods of use thereof are disclosed.

Claims

1. A method for the production of functional, non-tumorigenic, self-renewing, multipotent endodermal progenitor (EP) cells comprising, contacting stem cells with Activin A in serum free differentiation medium thereby inducing formation of definite endoderm (DE) cells;

and culturing DE cells in the presence of BMP4, VEGF, bFGF, and EGF or homologs thereof, wherein said EP cells are grown in spinner flask culture, comprising;

a) embedding a mixed culture of said stem cells and mouse embryonic fibroblasts (MEFs) in a biocompatible matrix;

b) adding said stem cell containing matrix to warmed EP culture medium, said EP medium being serum free differentiation medium comprising Activin A in an amount effective to induce formation of definite endoderm (DE) cells;

c) culturing DE cells in a spinner flask and culturing DE cells in the presence of a TGF-beta inducing agent, under conditions suitable for rafts of matrix containing EP cells to form, said culture being non-adherent;

said EP cells maintaining expression of EP cells markers and normal EP cell morphology; and

d) optionally harvesting said EP cells for further treatment or culture and/or freezing said EP cells obtained from step c).

4. The method of claim 1, wherein said biocompatible matrix is matrigel.

5. The method of claim 1, wherein said EP cells can be expanded 100-million fold in 13-15 passages in a 125 ml spinner flask.

6. The method of claim 1, wherein 150 million EP cells are generated from a 300 ml working cell culture volume after two passages in a 500 ml spinner flask.

2. The method of 1, wherein said cells express SOX17, FOXA2, and HNF4A and lack OCT4 or NANOG.

3. The method of claim 2, wherein said EP cells are induced to differentiate into endodermal tissues.

7. A method for the production of functional, non-tumorigenic, self-renewing, multipotent endodermal progenitor (EP) cells comprising, contacting stem cells with Activin A in serum free differentiation medium thereby inducing formation of definite endoderm (DE) cells;

and culturing DE cells in the presence of BMP4, VEGF, bFGF, and EGF or homologs thereof in liquid media or biocompatible matrix and optionally a fibroblast feeder layer;

thereby producing EP cells, wherein said media or biocompatible matrix further comprises TGF-beta or a TGF-beta inducing agent.