Differentiation of human embryonic stem cells (hESCs) to specific functional cell types can be achieved using methods that mimic in vivo embryonic developmental programs. Current protocols for generating hepatocytes from hESCs are hampered by inefficient differentiation procedures that lead to low yields and large cellular heterogeneity. We report here a robust and highly efficient process for the generation of high-purity (70%) hepatocyte cultures from hESCs that parallels sequential hepatic development in vivo. Highly enriched populations of definitive endoderm were generated from hESCs and then induced to differentiate along the hepatic lineage by the sequential addition of inducing factors implicated in physiological hepatogenesis.The differentiation process was largely uniform, with cell cultures progressively expressing increasing numbers of hepatic lineage markers, including GATA4, HNF4␣, ␣-fetoprotein, CD26, albumin, ␣-1-antitrypsin, Cyp7A1, and Cyp3A4. The hepatocytes exhibited functional hepatic characteristics, such as glycogen storage, indocyanine green uptake and release, and albumin secretion. In a mouse model of acute liver injury, the hESC-derived definitive endoderm differentiated into hepatocytes and repopulated the damaged liver. The methodology described here represents a significant step toward the efficient generation of hepatocytes for use in regenerative medicine and drug discovery.

During a screen to identify c-Jun activators, we isolated a cysteine protease, SuPr-1, that induced c-Jun-dependent transcription independently of c-Jun phosphorylation. SuPr-1 is a member of a new family of proteases that hydrolyze the ubiquitin-like modifier, SUMO-1. SuPr-1 hydrolyzed SUMO-1-modified forms of the promyelocytic leukemia gene product, PML, and altered the subcellular distribution of PML in nuclear PODs (PML oncogenic domains). SuPr-1 also altered the distribution of other nuclear POD-associated proteins, such as CBP and Daxx, that act as transcriptional regulators. SuPr-1 action on transcription was enhanced by PML, and SuPr-1 failed to activate transcription in PML-deficient fibroblasts. Our studies establish an important role for SUMO proteases in transcription.

Mitogen-activated protein (MAP) kinasesRaf-1, pp60O, and p2lm all play important roles in the transfer of signals from the cell surface to the nucleus. We have used the baculovirus/Sf9 insect cell system to elucidate the regulatory relationships between pp60v4vc, p21v-ru ¶ MAP kinase (p44erkl/IaPk), and Raf-1. In Sf9 cells, p44ekl/maPk iS activated by coexpression with either v-Raf or a constitutively activated form of Raf-1 (Raf22W The mechanism of activation of the cytoplasmic serinethreonine kinases p44erkl/mapk and p42erk2/mapk is clearly of interest. In response to extracellular stimuli, p44erkl/maPk and p42erk2/maPk undergo rapid phosphorylation on threonine and tyrosine residues and retardation in gel mobility. Activation of p44erkl/mapk and p42erk2/maPk requires phosphorylation of these proteins on both tyrosine and threonine residues (21, 22). These tyrosine and threonine residues, Thr-183 and Tyr-185 in p42erk2/maPk, are conserved in both isoforms, p44erkl/mapk and p42erk2/mapk. They are the only known cytoplasmic serine-threonine kinases known to be activated by tyrosine phosphorylation. Cytoplasmic MAP kinase activating factors have been identified (23, 24), purified to homogeneity (7, 25, 26), and recently cloned (27). In vitro, activated Raf-1 or v-raf can activate partially purified preparations of MAP kinase activator (14-16).Raf-1 and p44erkl/mapk both appear to be regulated by p21lms and by membrane tyrosine kinases (10,11,28,29). Stimulation of PC12 cells by nerve growth factor or epidermal growth factor activates p44erkl/mapk in these cells. In fact, expression of activated p21lms in PC12 cells is sufficient to activate p44erkl/mapk partially and to cause hyperphosphorylation and retardation in gel mobility of Raf-1 (10, 11). Moreover, expression of a dominant interfering allele of p21c-ms is sufficient to block nerve growth factor-induced p44erkl/mapk activation and hyperphosphorylation of 11).Since both Raf-1 and p44erkl/mapk are regulated by p21V-Ms and by membrane tyrosine kinases, we have used baculovirusencoded p2lv-ras, pp60v-src, p44erkl/mapk, and Raf-1 to examine the effects of each of the activated kinases on the activity of the other. As reported here and by others, p44erkl/mapk is activated by coexpression with either v-Raf or a constitutively activated form of Raf-1 (Raf22W). We report, however, that only a limited increase in the kinase activity of p44erkl/mapk iS seen after coexpression with either Raf-1 or with p21v-ms. The kinase activity of p44erkl/mapk iS greatly enhanced by coexAbbreviations: MAP, mitogen-activated protein; MBP, myelin basic protein.iTo whom reprint requests should be addressed. 5772

The antigen binding specificity of an antibody molecule is determined by the amino acid sequences of the variable (V) 1 regions of the heavy (H) and light (L) chains. The antibody repertoire of an individual is large and complex. The studies of antibody polypeptides and genes in mice have suggested three sources of diversity: multiple germline V genes (1-4), rearrangement of three sets of H chain (VH, D ]diversity], and JH [joining region of the H chain]) and two sets of L chain (VL andJL) germline segments (5-7); and two types of somatic alterations (7-9 and 10-12). Several questions, however, remain to be answered: (a) What are the relative contributions of the germline, combinatorial, and somatic variation mechanisms to the functional repertoire of antibody molecules? (b) Is somatic diversification a random process happening at the base somatic mutation rate, or is it a consequence of a V gene-specific mutation mechanism (13, 14)? (c) At what stage of B cell differentiation does somatic mutation take place?A better understanding of this problem requires a more detailed study of the heterogeneity of the response to specific antigens. As amino acid sequences of antibodies (15, 16) and nucleotide sequences (2, 11, 17, 18) of VH genes of restricted heterogeneity (idiotypes) have been obtained, the notion has emerged that somatic point mutation and combinatorial diversity of a single germline VH gene are responsible for the diversity observed among idiotype-crossreactive-antibody-producing cells.We have undertaken a systematic analysis of the immune response to the hapten 4-(hydroxy-3-nitrophenyl)acetyl (NP) at early stages of the response, using nucleotide sequence analysis of expressed V region genes.

The concept of reprogramming a cell is very intriguing and has immense therapeutic potential. Examples from physiology and developmental biology suggest that it may well be possible. Experimental approaches are beginning to suggest this also, in particular the initially astonishing accomplishment of somatic cell nuclear transfer and cloning. This chapter reviews current strategies and describes emerging methods for the proposition of reprogramming cells with cell extracts.