Amir Eden

Induced neuronal differentiation of human embryonic stem cells

Human embryonic stem (ES) cells are pluripotent cells capable of forming differentiated embryoid bodies (EBs) in culture. We examined the ability of growth factors under controlled conditions to increase the number of human ES cell-derived neurons. Retinoic acid (RA) and nerve growth factor (betaNGF) were found to be potent enhancers of neuronal differentiation, eliciting extensive outgrowth of processes and the expression of neuron-specific molecules. Our findings show that human ES cells have great potential to become an unlimited cell source for neurons in culture. These cells may then be used in transplantation therapies for neural pathologies.

Clone‐ and Gene‐Specific Aberrations of Parental Imprinting in Human Induced Pluripotent Stem Cells

Genomic imprinting is an epigenetic phenomenon whereby genes are expressed in a monoallelic manner, which is inherited either maternally or paternally. Expression of imprinted genes has been examined in human embryonic stem (ES) cells, and the cells show a substantial degree of genomic imprinting stability. Recently, human somatic cells were reprogrammed to a pluripotent state using various defined factors. These induced pluripotent stem (iPS) cells are thought to have a great potential for studying genetic diseases and to be a source of patient‐specific stem cells. Thus, studying the expression of imprinted genes in these cells is important. We examined the allelic expression of various imprinted genes in several iPS cell lines and found polymorphisms in four genes. After analyzing parent‐specific expression of these genes, we observed overall normal monoallelic expression in the iPS cell lines. However, we found biallelic expression of the H19 gene in one iPS cell line and biallelic expression of the KCNQ10T1 gene in another iPS cell line. We further analyzed the DNA methylation levels of the promoter region of the H19 gene and found that the cell line that showed biallelic expression had undergone extensive DNA demethylation. Additionally we studied the imprinting gene expression pattern of multiple human iPS cell lines via DNA microarray analyses and divided the pattern of expression into three groups: (a) genes that showed significantly stable levels of expression in iPS cells, (b) genes that showed a substantial degree of variability in expression in both human ES and iPS cells, and (c) genes that showed aberrant expression levels in some human iPS cell lines, as compared with human ES cells. In general, iPS cells have a rather stable expression of their imprinted genes. However, we found a significant number of cell lines with abnormal expression of imprinted genes, and thus we believe that imprinted genes should be examined for each cell line if it is to be used for studying genetic diseases or for the purpose of regenerative medicine. STEM CELLS 2009;27:2686–2690

Two yeast homologs of ECA39, a target for c-Myc regulation, code for cytosolic and mitochondrial branched-chain amino acid aminotransferases.

ECA39 was isolated as a target gene for c-Myc regulation in mice. We identified two homologs for the murine ECA39 in the yeast Saccharomyces cerevisiae, ECA39 and ECA40, as well as two human homologs. These genes show a significant homology to prokaryotic branched-chain amino acid aminotransferase (BCAT) (EC). To understand the function of eukaryotic ECA39 and ECA40, we deleted either gene from the yeast genome. Activity of branched-chain amino acid aminotransferase was measured in the wild-type and mutants with either leucine, isoleucine, or valine as substrates. The results demonstrate that in S. cerevisiae ECA39 and ECA40 code for mitochondrial and cytosolic branched-chain amino acid aminotransferases, respectively. ECA39 is highly expressed during log phase and is down-regulated during the stationary phase of growth, while ECA40 shows an inverse pattern of gene expression. In agreement with these results, while we previously showed that deletion of ECA39 affected the cell cycle in proliferating cells, we do not observe a growth phenotype in eca40Δ cells. We suggest that BCAT is a target for c-Myc activity and discuss the evolutionary conservation of prokaryotic and eukaryotic BCATs and their possible involvement in regulation of cell proliferation.

Glucose regulates cyclin D2 expression in quiescent and replicating pancreatic β-cells through glycolysis and calcium channels.

Understanding the molecular triggers of pancreatic β-cell proliferation may facilitate the development of regenerative therapies for diabetes. Genetic studies have demonstrated an important role for cyclin D2 in β-cell proliferation and mass homeostasis, but its specific function in β-cell division and mechanism of regulation remain unclear. Here, we report that cyclin D2 is present at high levels in the nucleus of quiescent β-cells in vivo. The major regulator of cyclin D2 expression is glucose, acting via glycolysis and calcium channels in the β-cell to control cyclin D2 mRNA levels. Furthermore, cyclin D2 mRNA is down-regulated during S-G(2)-M phases of each β-cell division, via a mechanism that is also affected by glucose metabolism. Thus, glucose metabolism maintains high levels of nuclear cyclin D2 in quiescent β-cells and modulates the down-regulation of cyclin D2 in replicating β-cells. These data challenge the standard model for regulation of cyclin D2 during the cell division cycle and suggest cyclin D2 as a molecular link between glucose levels and β-cell replication.

Involvement of branched-chain amino acid aminotransferase (Bcat1/Eca39) in apoptosis

The branched‐chain amino acid aminotransferase, Bcat1/Eca39, catalyzes the first step of branched‐chain amino acid catabolism. Bcat1/Eca39 was originally isolated from a c‐myc‐induced tumor and was proven to be a direct target for c‐Myc regulation. The gene is highly conserved in evolution and disruption of its yeast homolog affects cell growth. To assess the role of Bcat1/Eca39 in mammalian cells, we overexpressed Bcat1/Eca39 in murine cells and studied effects on cell growth. Overexpression of Bcat1/Eca39 had no apparent effect on the proliferation of cells grown with high serum concentrations, but under serum deprivation conditions, led to a decrease in cell viability. Cell death under these conditions displayed apoptotic features. The branched‐chain keto acid, α‐ketoisocaproate, a metabolite of leucine catabolism produced by BCAT1/ECA39, was previously found to inhibit cell growth. We show that α‐ketoisocaproate can induce rapid apoptotic cell death. This observation suggests that the growth inhibitory effect of BCAT1/ECA39 and its apoptosis promoting effect may be mediated by the levels of the products of BCAT1/ECA39 activity, namely, branched‐chain keto acids.

A Transgenic Mouse Marking Live Replicating Cells Reveals In Vivo Transcriptional Program of Proliferation

Most adult mammalian tissues are quiescent, with rare cell divisions serving to maintain homeostasis. At present, the isolation and study of replicating cells from their in vivo niche typically involves immunostaining for intracellular markers of proliferation, causing the loss of sensitive biological material. We describe a transgenic mouse strain, expressing a CyclinB1-GFP fusion reporter, that marks replicating cells in the S/G2/M phases of the cell cycle. Using flow cytometry, we isolate live replicating cells from the liver and compare their transcriptome to that of quiescent cells to reveal gene expression programs associated with cell proliferation in vivo. We find that replicating hepatocytes have reduced expression of genes characteristic of liver differentiation. This reporter system provides a powerful platform for gene expression and metabolic and functional studies of replicating cells in their in vivo niche.

Aberrant Epigenetic Silencing of Tumor Suppressor Genes Is Reversed by Direct Reprogramming

Direct reprogramming procedures reset the epigenetic memory of cells and convert differentiated somatic cells into pluripotent stem cells. In addition to epigenetic memory of cell identity, which is established during development, somatic cells can accumulate abnormal epigenetic changes that can contribute to pathological conditions. Aberrant promoter hypermethylation and epigenetic silencing of tumor suppressor genes (TSGs) are now recognized as an important mechanism in tumor initiation and progression. Here, we have studied the fate of the silenced TSGs p16(CDKN2A) during direct reprogramming. We find that following reprogramming, p16 expression is restored and is stably maintained even when cells are induced to differentiate. Large‐scale methylation profiling of donor cells identified aberrant methylation at hundreds of additional sites. Methylation at many, but not all these sites was reversed following reprogramming. Our results suggest that reprogramming approaches may be applied to repair the epigenetic lesions associated with cancer. STEM CELLS 2010;28:1349–1354

Synergism between DNA methylation and macroH2A1 occupancy in epigenetic silencing of the tumor suppressor gene p16(CDKN2A)

Promoter hypermethylation and heterochromatinization is a frequent event leading to gene inactivation and tumorigenesis. At the molecular level, inactivation of tumor suppressor genes in cancer has many similarities to the inactive X chromosome in female cells and is defined and maintained by DNA methylation and characteristic histone modifications. In addition, the inactive-X is marked by the histone macroH2A, a variant of H2A with a large non-histone region of unknown function. Studying tumor suppressor genes (TSGs) silenced in cancer cell lines, we find that when active, these promoters are associated with H2A.Z but become enriched for macroH2A1 once silenced. Knockdown of macroH2A1 was not sufficient for reactivation of silenced genes. However, when combined with DNA demethylation, macroH2A1 deficiency significantly enhanced reactivation of the tumor suppressor genes p16, MLH1 and Timp3 and inhibited cell proliferation. Our findings link macroH2A1 to heterochromatin of epigenetically silenced cancer genes and indicate synergism between macroH2A1 and DNA methylation in maintenance of the silenced state.

The Genetic Program of Pancreatic β-Cell Replication In Vivo.

The molecular program underlying infrequent replication of pancreatic β-cells remains largely inaccessible. Using transgenic mice expressing green fluorescent protein in cycling cells, we sorted live, replicating β-cells and determined their transcriptome. Replicating β-cells upregulate hundreds of proliferation-related genes, along with many novel putative cell cycle components. Strikingly, genes involved in β-cell functions, namely, glucose sensing and insulin secretion, were repressed. Further studies using single-molecule RNA in situ hybridization revealed that in fact, replicating β-cells double the amount of RNA for most genes, but this upregulation excludes genes involved in β-cell function. These data suggest that the quiescence-proliferation transition involves global amplification of gene expression, except for a subset of tissue-specific genes, which are “left behind” and whose relative mRNA amount decreases. Our work provides a unique resource for the study of replicating β-cells in vivo.

CHARACTERIZATION OF A BRANCHED-CHAIN AMINO-ACID AMINOTRANSFERASE FROM SCHIZOSACCHAROMYCES POMBE

The Saccharomyces cerevisiae genes for the cytosolic and mitochondrial branched‐chain amino‐acid aminotransferases (BCAT) were isolated recently. These genes show significant homology to mammalian ECA39, originally isolated as a gene regulated by the c‐myc oncogene. We now report the isolation of the Schizosaccharomyces pombe eca39/BCAT gene. The S. pombe protein shows 47–52% identity to other eukaryotic BCAT proteins isolated from S. cerevisiae, nematode, mouse and man. A genetic growth assay for BCAT activity was established using an S. cerevisiae strain disrupted in both BCAT isoenzymes. Consequently, the activity of the S. pombe BCAT was demonstrated by genetic and biochemical means. Possible applications of BCAT‐encoding genes as selection markers in yeast transformation are proposed. The sequence has been deposited in the GenBank data library under Accession Number U88029.