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Hepatocyte lineage cells derived from pluripotent stem cells

A technology of pluripotent stem cells and human pluripotent stem cells, applied in the field of directed differentiation, can solve the problems of mouse embryonic cells not responding to the same culture conditions and pluripotent cells being fragile

Inactive Publication Date: 2003-08-27
GERON CORPORATION
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, monkey and human pluripotent cells proved to be much more fragile and did not respond to the same culture conditions as mouse embryonic cells

Method used

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  • Hepatocyte lineage cells derived from pluripotent stem cells
  • Hepatocyte lineage cells derived from pluripotent stem cells
  • Hepatocyte lineage cells derived from pluripotent stem cells

Examples

Experimental program
Comparison scheme
Effect test

preparation example Construction

[0064] Preparation of primate pluripotent stem cells (pPS)

[0065] Embryonic stem cells can be isolated from fibroblasts of members of the primate species (Thomson et al., Proc. Natl. Acad. Sci. USA 92:7844, 1995). Human embryonic stem cells (hES) can be prepared from human fibroblasts using the techniques described by Thomson et al.

[0066] To obtain human fibroblasts, embryos preimplanted in vivo or in vitro fertilized (IVF) can be used, or single-cell human embryos can be expanded to the blastocyst stage (Bongso et al., Hum Reprod 4:706, 1989). Briefly, human embryos were grown to the blastocyst stage in G1.2 and G2.2 media (Gardner et al., Fertil. Steril. 69:84, 1998). Developed blastocysts can be selected for isolation of ES cells. The zona pellucida was removed from blastocysts by brief exposure to pronase (Sigma). The inner cell mass was separated by immunosurgery, in which blastocysts were exposed to 1:50 diluted rabbit anti-human splenocyte antiserum for 30 minut...

Embodiment 1

[0187] Example 1: Differentiation of human embryonic stem cells with n-butyrate

[0188] Embryoid bodies (EBs) were prepared as described in the previous section. After 5 days of suspension culture, they were harvested and plated on growth factor-reduced Matrigel(R) coated plates, and culture tank slides (Nunc). The following three conditions were used in parallel:

[0189] A culture medium containing 20% ​​fetal bovine serum (FBS);

[0190] - Medium containing 20% ​​FBS and 5 mM sodium butyrate (Sigma);

[0191] • Contains 20% FBS, 0.5% DMSO (ATCC), 4 μm dexamethasone (Sigma), 150 ng / ml insulin, 10 ng / ml EGF, 600 nM glucagon (Sigma).

[0192] In each case, medium was changed daily and cells were fixed for immunocytochemistry on day 4 after plating.

[0193] 1 day after plating, EBs plated alone in 20% FBS appeared healthy, they were almost all attached to the plate and appeared to be proliferating. After a few days, only the cells in FBS survived well and differentiated ...

Embodiment 2

[0199] Example 2: Markers Expressed by Differentiated Cells

[0200] After 4 or 5 days of suspension culture, the embryoid bodies derived from hES cells were collected and plated on Matrigel®-coated 6-well plates (for RNA extraction) and culture tank slides (for immunocytochemistry) containing 20 % FBS and 5 mM sodium n-butyrate in the medium. Change the medium every day or every other day. Many cells die on day 1, then fewer cells die in the following days.

[0201] figure 2 Morphology of differentiated cells after 6 days of culture with n-butyrate is shown. The same culture shows 6 different fields (1Ox on top, 2Ox in other columns). The cells are remarkably homogeneous, displaying the large polygonal surface and binucleated center characteristic of mature hepatocytes.

[0202]On day 6 after plating in differentiating agents, cells were analyzed for marker expression by RT-PCR and immunocytochemistry according to the methods described above. Glycogen content in these ...

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Abstract

It has been found that when pluripotent stem cells are cultured in the presence of hepatocyte differentiating agents, a population of cells is derived that has a remarkably high proportion of cells with a stem cell phenotype. In one example, human embryonic stem cells are allowed to form embryoid bodies and then mixed with the differentiation agent n-butyrate, optionally supplemented with maturation factors. In another example, n-butyrate is added to human embryonic stem cells in feeder-free culture. A remarkably homogeneous population of cells was obtained in both methods, consisting primarily of cells with morphological characteristics of hepatocytes, expressing characteristic surface markers of hepatocytes, and also possessing enzymatic and biosynthetic roles important for liver function. Since hepatocytes readily proliferate in culture, this system provides a rich source of hepatocyte lineage cells for various uses, such as drug screening and supplementation of liver function in clinical therapy.

Description

field of invention [0001] The present invention relates generally to the field of cell biology of embryonic cells and hepatocytes. More specifically, the present invention relates to the directed differentiation of human pluripotent stem cells towards cells of the hepatocyte lineage under specific culture conditions. [0002] References to related applications [0003] This application claims priority to US Provisional Patent Application 60 / 200,095, filed April 27, 2000; and US Utility Model Application 09 / 718,308, filed November 20, 2000. The priority application is hereby incorporated by reference in its entirety for enforcement in the United States. Background of the invention [0004] Liver disease affects millions of people around the world. Fulminant liver failure is the clinical term for the rapid and catastrophic cessation of liver function, usually resulting in death within hours. Other forms of liver disease, such as chronic hepatitis and cirrhosis, involve ins...

Claims

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Application Information

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IPC IPC(8): A61L27/00A61P1/16C12N5/02C12N15/09C12N5/071C12N5/10C12Q1/02
CPCC12N2500/30C12N2501/115C12N2503/02C12N2501/39C12N5/067C12N2501/33C12N2501/11C12N2506/02C12N2533/90C12N2501/12C12N2501/385C12N2501/335C12N2501/148C12N2500/36A61P1/16C12N5/0602
Inventor 拉克舒美·蓝哈拉莫莉莎·K·卡本特
Owner GERON CORPORATION
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