Novel population of hepatocytes derived via definitive endoderm (de-hep) from human blastocysts stem cells

a technology of endoderm and blastocysts, which is applied in the field of new hepatocyte population derived via definitive endoderm (dehep) from human blastocysts stem cells, can solve the problems of large death rate, limited efficacy of this procedure, and major burden on the health care system, and achieves the effect of increasing the percentag

a technology of endoderm and blastocysts, which is applied in the field of new hepatocyte population derived via definitive endoderm (dehep) from human blastocysts stem cells, can solve the problems of large death rate, limited efficacy of this procedure, and major burden on the health care system, and achieves the effect of increasing the percentag

US20100190202A1Inactive Publication Date: 2010-07-29CELLARTIS AB (SE)
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  • Novel population of hepatocytes derived via definitive endoderm (de-hep) from human blastocysts stem cells
  • Novel population of hepatocytes derived via definitive endoderm (de-hep) from human blastocysts stem cells
  • Novel population of hepatocytes derived via definitive endoderm (de-hep) from human blastocysts stem cells

Examples

Experimental program
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Effect test

example 1

Starting Material

[0220]The starting material for the present invention is suitably pluripotent undifferentiated hBS cells, such as undifferentiated hBS cell lines. Such material can be obtained from Cellartis AB, www.cellartis.com. Cellartis AB is the largest source of defined hBS cell lines world wide and has more than 30 hBS cell lines and subclones available today. Two of the cell lines are listed in the National Institutes of Health (NIH) Human Embryonic Stem Cell Registry, http: / / stemcells.nih.gov / research / registry / and 20 in the UK Stem Cell Bank. Additionally, Cellartis can provide hBS cell lines approved for research use by major markets such as Japan, Germany and France. For the present invention hBS cell line SA002, SA002.5, SA034, SA167, SA181, SA348 and SA461 were used specifically. Characteristics of the hBS cells recommended as starting material are the following: positive for alkaline phosphatase, SSEA-3, SSEA-4, TRA 1-60, TRA 1-81, Oct-4, negative for SSEA-1, telomer...

example 2

Inducing Undifferentiated hBS Cells into DE and Further into Functional Hepatocytes

[0224]Undifferentiated hBS cells were maintained and propagated in vitro. At day 4-7 after passage undifferentiated hBS cell colonies were treated according to a basic differentiation program. Each step in this protocol is characterized by key components that are crucial;

Stage I, Definitive endoderm (DE): low serum and Activin A

Stage II, DE-Hep progenitor cells: members of the TGFbeta superfamily of proteins, especially BMP4

Stage III, functional DE-Hep cells: HGF

Changes in growth factor exposure time, concentration, various combinations of them and length of treatment have been tested

[0225]An overview of the basic method and variations is presented in FIG. 1a. The undifferentiated hBS cells are induced to DE by incubation with Activin A (100 ng / ml) for 3-5 days in RPMI Advanced medium or DMEM medium or a comparable medium (stage I, day 0). The medium is changed every day. The first day of Activin A in...

example 3

Morphology of Definitive Endoderm, Stage I

[0265]Activin A treatment was associated to morphological changes and distinct differentiation pattern of the colonies. After 3-5 days, treated by an Activin A-containing medium, cells gathered together and formed a ring of epithelial cells around the colony (FIG. 2). Those homogenous growing cells expanded over the plate and could be passaged to a Mouse feeder layer containing plate.

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Abstract

The present disclosure relates to a novel hepatocyte-like cell progenitor and / or a novel hepatocyte-like cell derived via definitive endoderm from human blastocyst-derived stem (hBS) cells, to a method for the preparation of such cells and to the potential use of such cells in, e.g., pharmaceutical drug discovery and development, toxicity testing, cell therapy and medical treatment. In particular is presented a definitive endoderm derived hepatocyte-like cell with important liver-expressed marker genes and important metabolizing enzymes, as well as drug transporters.

Description

FIELD OF THE INVENTION[0001]The present invention relates to a novel hepatoctye-like cell population derived via definitive endoderm from human blastocyst-derived stem (hBS) cells, to a method for the preparation of such cells and to the potential use of such cells in e.g. pharmaceutical drug discovery and development, toxicity testing, cell therapy and medical treatment. The cells are denoted DE-hep cells herein.[0002]Furthermore, the DE-Hep cells exhibits properties of functional human hepatocytes which make them attractive for use in in vitro studies of hepatogenesis such as early liver development processes or liver-regenerative disorders or malformations.BACKGROUND OF THE INVENTION[0003]Pluripotent human stem cells are expected to revolutionize the accessibility to a variety of human cell types. The possibility to propagate pluripotent human blastocyst-derived stem (hBS) cells and subsequently differentiate them into the desired target cell types will provide a stable and virtu...

Claims

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Application Information

Patent Timeline
29 Jul 2010
Publication
US20100190202A1
IPC
C12Q1/02; C12N5/071
CPC
C12N5/067; C12N2501/113; C12N2501/115; C12N2501/12; C12N2506/02; C12N2501/16; C12N2501/237; C12N2501/39
Inventors
HEINS, NICO; BROLEN, GABRIELLA