Lineage restriction of human hepatic stem cells to mature fates is made efficient by tissue-specific biomatrix scaffolds.

Unlabelled: 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.

Culture of mouse embryonic stem cells.

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.

Role of the avian retrovirus mRNA leader in expression: evidence for novel translational control.

Avian retroviral mRNAs contain a long 5' untranslated leader of approximately 380 nucleotides. The leader includes sequences required for viral replication and three AUG codons which precede the AUG codon used for translational initiation of the gag and env genes. We have used sensitive, quantitative assays of viral gene transcription and translation to analyze the role of this mRNA leader in viral gene expression.

Use of avian retroviral-bovine growth hormone DNA recombinants to direct expression of biologically active growth hormone by cultured fibroblasts.

A variety of recombinant DNA molecules were constructed in which an avian retroviral long terminal repeat (LTR) was ligated to the bovine growth hormone (bGH) gene. The retroviral LTR was derived from a plasmid clone of a Schmidt Ruppin B strain of Rous sarcoma virus while the bGH gene was subcloned from a lambda bacteriophage genomic library. Using a transient eukaryotic expression assay system, recombinant plasmid constructs were screened for their ability to direct expression and secretion of bGH.

Nucleotide sequence of the long terminal repeat and flanking cellular sequences of avian endogenous retrovirus ev-2: variation in Rous-associated virus-0 expression cannot be explained by differences in primary sequence.

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.