Generation of hepatocytes from cultured mouse embryonic stem cells

Abstract

Embryonic stem (ES) cells are pluripotent cells derived from the inner cell mass of fertilized blastocysts in vitro. ES cells can be induced to undergo differentiation into potentially all cell types. The aim of this study is to examine the differentiating potential of mouse ES cells into hepatocytes in the presence of retinoic acid (RA), hepatocyte growth factor (HGF), and ␤-nerve growth factor (␤-NGF). RA, HGF, and ␤-NGF were added to the cell culture. Hepatocyte induction was confirmed morphologically, as well as biochemically, through immunohistochemical assays of ␣ 1 -antitrypsin (␣ 1 -AT) and alfafetaprotein (AFP) expression and reverse-transcriptase polymerase chain reaction tests for the presence of albumin, transthyretin, glucose 6 phosphates, hepatic nuclear factor 4, and SAPK/ERK kinase-1 (SEK1) messenger RNA, produced only by functioning hepatocytes. Fifteen days after the addition of HGF and ␤-NGF to the cell culture, many epithelioid cells were noticed. ␣ 1 -AT, AFP, albumin, transthyretin, glucose 6 phosphates, hepatic nuclear factor 4, and SEK1 messenger RNA expression also was detected, indicating successful ES cell differentiation into functioning hepatocytes. However, in the presence of RA alone, only transthyretin messenger RNA was positive, whereas no other expression pertaining to functioning hepatocytes could be detected. In the presence of HGF and ␤-NGF, mouse ES cells can differentiate into functioning hepatocytes, whereas RA function is limited. E mbryonic stem (ES) cells are continuously growing cell lines derived from the inner cell mass of 3.5-day blastocysts. Under proper experimental conditions in vitro, ES cells have full developmental potential to differentiate into cell types of all three germ layers. 1,2 In addition, when allowed to form three-dimensional structures known as embryoid bodies (EBs), ES cells initiate the progenitor from a continuously growing stem-cell population in vitro, providing a unique system for the analysis of both cellular and molecular events involved in the first stage of lineage determination. ES cells also have been used to study the differentiation of various cell types and tissues in vitro, such as neural cells, 3 hematopoietic lineages, and cardiomyocytes. 4 ES-derived cells have been transplanted successfully into the neuronal system and liver of fetal and adult mice, where they show integration of morphological characteristics and function. 5 Because of the shortage of liver donors, hepatocyte transplantation currently is being evaluated as a potential method of providing metabolic support during acute and chronic liver failure and can be used to compensate specific liver functions in patients with inherited metabolic liver diseases. 6 Hepatocytes currently have not been considered an ideal source for cell transplantation. We wanted to know whether ES cells could be a new cell source for cell transplantation to treat liver diseases.The aim of our study is to investigate the effects of retinoic acid (RA), hepatocyte growth factor (HGF), and ␤-ner...

Hepatic precursors derived from murine embryonic stem cells contribute to regeneration of injured liver

“…10 The in vitro approaches involve the formation of embryoid bodies (EBs) to re-create the inductive microenvironment required for liver organogenesis 11,12 or treatment with specific growth factors and cytokines critical for hepatocyte differentiation such as hepatocyte growth factor, fibroblast growth factor, and oncostatin. 1,13 Also, the introduction of genes promoting endodermal differentiation into ES cells, 14 modification of culture microenvironment by supplementation with extracellular matrix proteins or coculture with other cell types, 15,16 is used to direct ES cells toward a hepatocytic lineage. Nevertheless, these strategies generally have been inefficient, producing small numbers of hepatocyte precursors within heterogeneous cell populations.…”

Directed hepatic differentiation from embryonic stem cells

“…ESC will differentiate and form an EB after removing feeder cells and culturing in suspension culture medium without leukemia inhibitory factor (LIF) or FGF2; The EBs will spontaneously differentiate into different cell types of the three germ layers including liver cells in adherent culture conditions (Chinzei et al, 2002;Kuai et al, 2003;Asahina et al, 2004;Kumashiro et al, 2005). In order to avoid spontaneous differentiation of ESC forming a mixture of many types of cells, researchers have successfully induced embryonic stem cells to differentiate into liver cells through the reconstruction of an appropriate in vivo microenvironment.…”

Stem cell plasticity: Learning from hepatogenic differentiation strategies

“…In addition, until day 30, no tumors were detected, and fibrosis decreased. Kuai et al (2003) reported that the nerve growth factor (NGF)-␤ also promotes hepatic differentiation, which is increased in the presence of HGF and RA. Miyashita et al (2002) also demonstrated in vitro hepatic differentiation through the formation of EBs without using hepatocyte-specific cytokines.…”