When differentiated in the presence of activin A in serum-free conditions, mouse embryonic stem cells efficiently generate an endoderm progenitor population defined by the coexpression of either Brachyury, Foxa2 and c-Kit, or c-Kit and Cxcr4. Specification of these progenitors with bone morphogenetic protein-4 in combination with basic fibroblast growth factor and activin A results in the development of hepatic populations highly enriched (45-70%) for cells that express the alpha-fetoprotein and albumin proteins. These cells also express transcripts of Afp, Alb1, Tat, Cps1, Cyp7a1 and Cyp3a11; they secrete albumin, store glycogen, show ultrastructural characteristics of mature hepatocytes, and are able to integrate into and proliferate in injured livers in vivo and mature into hepatocytes expressing dipeptidyl peptidase IV or fumarylacetoacetate hydrolase. Together, these findings establish a developmental pathway in embryonic stem cell differentiation cultures that leads to efficient generation of cells with an immature hepatocytic phenotype.

Embryonic stem (ES) cells are revolutionizing the field of developmental biology as a potential tool to understand the molecular mechanisms occurring during the process of differentiation from the embryonic stage to the adult phenotype. ES cells harvested from the inner cell mass (ICM) of the early embryo can proliferate indefinitely in vitro while retaining the ability to differentiate into all somatic cells. Emerging results from mice models with ES cells are promising and raising tremendous hope among the scientific community for the ES-cell based cell replacement therapy (CRT) of various severe diseases. ES cells could potentially revolutionize medicine by providing an unlimited renewable source of cells capable of replacing or repairing tissues that have been damaged in almost all degenerative diseases such as diabetes, myocardial infarction and Parkinson's disease. This review updates the progress of ES cell research in CRT, discusses about the problems encountered in the practical utility of ES cells in CRT and evaluates how far this approach is successful experimentally.

Background & Aims Highly proliferative fetal liver stem/progenitor cells (FLSPC) repopulate livers of normal recipients by cell competition. We investigated the mechanisms by which FLSPC repopulate livers of older, compared with younger rats. Methods Fetal liver cells were transplanted from DPPIV+ F344 rats into DPPIV− rats of different ages (2, 6, 14, or 18 months); liver tissues were analyzed 6 months later. Cultured cells and liver tissues were analyzed by reverse transcription PCR, immunoblot, histochemistry, laser-capture microscopy, and TUNEL analyses. Results We observed 4–5-fold increases in liver repopulation when FLSPC were transplanted into older, compared with younger, rats. mRNA levels of cyclin-dependent kinase inhibitors increased progressively in livers of older rats; hepatocytes from 20-month old rats had 6.1-fold higher expression of p15INK4b and were less proliferative, in vitro, than hepatocytes from 2-month old rats. Expression of p15INK4b in cultured hepatocytes was upregulated by activin A, which increased in liver during aging. Activin A inhibited proliferation of adult hepatocytes, whereas FLSPC were unresponsive because they had reduced expression of activin receptors (e.g. ALK-4). In vivo, expanding cell clusters derived from transplanted FLSPC had lower levels of ALK-4 and p15INK4b and increased levels of Ki-67, compared with the host parenchema. Liver tissue of older rats had 3-fold more apoptotic cells than of younger rats. Conclusions FLSPC, resistant to activin A signaling, repopulate livers of older rats; hepatocytes in older rats have less proliferation, because of increased activin A and p15INK4b levels, and increased apoptosis than of younger rats. These factors and cell types might be manipulated to improve liver cell transplantation strategies in patients with liver diseases in which activin A levels are increased.

SummaryIn vitro differentiation of embryonic stem(ES) cells results in generation of tissue-specific somaticc ells and mayr epresent a powerful tool forg eneral understandingo fc ellular differentiation and development in vivo.Culturing of most ES cell lines requires murinee mbryonicf ibroblasts (MEF),w hich mayi nfluence adventitiously the genetic differentiation programofES cells.We compared the expression profile of keydevelopmental genesinthe MEF-independentCGR8EScellline and in the MEFdependentD 3E Sc elll ine.Using neomycin-resistant MEFs we demonstrated that MEFs areable to contaminate theD3EScells even after removing the MEFs.Subsequently, optimal differentiation conditions were establishedf or thed ifferentiation of CGR8 ES cells into various germ layerc ells.Detailed gene expression studies in differentiatingCGR8cells were donebyR TKeywords CGR8 embryonic stemc ells,g enee xpression,m icroarrays, development PCR analysis and by microarrayanalysis demonstrating ageneral trendofthe assessedgenes to be expressedeitherin3days-or 10-days oldembryoid bodies(EBs)whencompared to undifferentiatedE Sc ells.S ubsetsw ithin the various functional gene classesweredefinedthat arespecificallyup-or down-regulated in concert. Interestingly, the presentr esults demonstratet hat developmental processes towardgermlayer formation areirreversible andmostlyindependent of the cultureconditions. Notably, apoptotic and mitochondrial ribosomal genesweredownand up-regulated in 10-days oldEBs, respectively, whereas compared to the 3-days old EBs whereas theactivity of the extracellular signal-regulatedkinase(ERK)1/2 decreasedwith progressivedevelopment.This article defines aplatformfor ES cell differentiation and gene expression studies.