Current protocols for differentiation of stem cells make use of multiple treatments of soluble signals and/or matrix factors and result typically in partial differentiation to mature cells with under-or overexpression of adult tissue-specific genes. We developed a strategy for rapid and efficient differentiation of stem cells using substrata of biomatrix scaffolds, tissue-specific extracts enriched in extracellular matrix, and associated growth factors and cytokines, in combination with a serum-free, hormonally defined medium (HDM) tailored for the adult cell type of interest. Biomatrix scaffolds were prepared by a novel, four-step perfusion decellularization protocol using conditions designed to keep all collagen types insoluble. The scaffolds maintained native histology, patent vasculatures, and 1% of the tissue's proteins but >95% of its collagens, most of the tissue's collagen-associated matrix components, and physiological levels of matrix-bound growth factors and cytokines. Collagens increased from almost undetectable levels to >15% of the scaffold's proteins with the remainder including laminins, fibronectins, elastin, nidogen/entactin, proteoglycans, and matrix-bound cytokines and growth factors in patterns that correlate with histology. Human hepatic stem cells (hHpSCs), seeded onto liver biomatrix scaffolds and in an HDM tailored for adult liver cells, lost stem cell markers and differentiated to mature, functional parenchymal cells in 1 week, remaining viable and with stable mature cell phenotypes for more than 8 weeks. Conclusion: Biomatrix scaffolds can be used for biological and pharmaceutical studies of lineage-restricted stem cells, for maintenance of mature cells, and, in the future, for implantable, vascularized engineered tissues or organs. (HEPATOLOGY 2011;53:293-305) T he ongoing revolution in stem cell research has made possible the identification and isolation of stem cell populations including those from fetal and postnatal tissues.1 The potential of human hepatic stem cells (hHpSCs) and other stem/progenitors for pharmaceutical research, cell-based therapies,

NTRE 7 is an avian retrovirus recombinant of the endogenous nononcogenic Rous-associated virus-0 (RAV-0) and the oncogenic, exogenous, transformationdefective (td) Prague strain of Rous sarcoma virus B (td-PrRSV-B). Oligonucleotide mapping had shown that the recombinant virus is indistinguishable from its RAV-0 parent except for the 3'-end sequences, which were derived from tdPrRSV-B. However, the virus exhibits properties which are typical of an exogenous virus: it grows to high titers in tissue culture, and it is oncogenic in vivo. To accurately define the genetic region responsible for these properties, we determined the nucleotide sequences of the recombinant and its RAV-0 parent by using molecular clones of their DNA. These were compared with sequences already available for PrRSV-C, a virus closely related to the exogenous parent tdPrRSV-B. The results suggested that the crossover event which generated NTRE 7 took place in a region -501 to -401 nucleotides from the 3' end of the td-PrRSV parental genome and that sequences to the right of the recombination region were responsible for its growth properties and oncogenic potential. These sequences included a 148-base-pair exogenous-virus-specific region that was absent from the RAV-0 genome and the U3 region of the long terminal repeat. Since the exogenous-virus-specific sequences are expected to be missing from transformation-defective mutants of the Schmidt-Ruppin strain of RSV, which, like other exogenous viruses, grow to high titers in tissue culture and are oncogenic in vivo, we concluded that the growth properties and oncogenic potential of the exogenous viruses are determined by sequences in the U3 region of the long terminal repeat. However, we propose that the exogenous-virus-specific region may play a role in determining the oncogenic spectrum of a given oncogenic virus.

In this unit standard culture conditions for mouse embryonic stem cells (mESCs) on primary murine embryonic fibroblast (PMEF or MEF) monolayers, culture conditions without MEF for feeder‐independent mESCs, and culture conditions in chemically defined media for both feeder‐independent mESCs and feeder‐dependent mESCs are described. For expansion of an mESC line, it is crucial that cells maintain their undifferentiated state and their self‐renewal capacity, and that they remain karyotypically normal, all of which are necessary for successful chimerization of the germ line upon blastocyst injection. Derivation and culture conditions for the original mESCs have been described (notably Robertson, 1987; Smith, 1991; Nagy et al., 2003), however, as there are more and more mESC lines available, it becomes evident that culture conditions are cell‐line specific to some extent, and there is a constant demand for culturing details for mESC lines derived from different mouse strains. Curr. Protoc. Stem Cell Biol. 5:1C.4.1‐1C.4.19. © 2008 by John Wiley & Sons, Inc.

The scanning hypothesis states that the initiation of translation of eucaryotic mRNAs involves binding of the 40S ribosomal subunit to the mRNA 5' terminus followed by migration along the RNA until an initiator AUG codon is encountered (i4). In the vast majority of eucaryotic mRNAs, the 40S subunit mnigrates to the 5'-proximal initiator AUG codon where translation begins. Initiator AUG codons are preceded by the consensus sequence GXXAUG; the only highly conserved nucleotide is a purine at position -3 from the AUG codon (15, 16). Initiator AUG codons which align with the major translational reading frame of mRNAs have been found between 3 and 572 nucleotides from the 5' terminus (16). A small percentage of cellular mRNAs and some viral mRNAs contain additional AUG codons which lie upstream of the major open reading frame encoded by the mRNA. These upstream AUGs have two major characteristics: (i) they are not usually preceded by consensus AUG initiation sequences, and (ii) they are followed closely by in-frame termination codons which precede the AUG start codon of the major protein reading frame. the riRNA, as has been suggested in the cases of both poliovirus (25) and ASV (5).Although several predictions of the scanning model of translation initiation have been substantiated by experimental data, several problems remain unresolved. In particular, it remains unclear whether the primary sequence or secondary structure (or both) of the mRNA leader is important in modulating mRNA translational efficiency. One approach to this question has been to alter the mRNA leader region of cloned genes which have been engineered into highefficiency expression vectors (11,18,21,23). The effect of manipulating the leader region can be measured by detection of the gene product after transfection of recipient cells. This approach has been useful in demonstrating that upstream initiator AUG codons can interfere with the initiation of downstream reading frames. However, this method is not ideal for determining the normal function of the 5' leader region for at least two reasons: (i) the 5' terminus of the mRNA is donated from the expression vector, thus generating a chimeric, artificial mRNA, and (ii) the host cell which has been engineered for high expression is not the normal environment for the expressed RNA.The model system we have chosen to examine the effect of the leader region on gene expression utilizes the ASV envelope (env) gene and offers several advantages. The transcription of env mRNA is directed by the natural retroviral long terminal repeat (LTR) promoter, thus generating a native mRNA. The assay is also performed using quail cells, a natural host for this virus. The most important advantage, however, is the existence of sensitive, quantitative assays for both env mRNA and env protein (2-4). To examine the effect of the viral leader on env mRNA expression, we have introduced both natural leader segments from related avian retroviruses and artificially generated leader deletion muta-372 on May 12, 2018 by guest

A fragment of chicken DNA containing the left long terminal repeat of endogenous retrovirus ev-2 and flanking cellular sequences has been molecularly cloned and analyzed. Comparison with sequence data from the analogous regions of ev-1 and Rous-associated virus-0 viral DNA reveals similarities among flanking regions of the integrated proviruses and among all three long terminal repeats. From the latter finding, we conclude that the difference in level of expression of ev-2 and its progeny Rous-associated virus-0 provirus cannot be due to sequence differences in their upstream long terminal repeats.