Richard Malavarca

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. 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.

NTRE 7 is an avian retrovirus recombinant of the endogenous nononcogenic Rous-associated virus-0 (RAV-0) and the oncogenic, exogenous, transformation-defective (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 td-PrRSV-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 td-PrRSV-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.

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. By substituting segments from related viruses, we had previously shown that the endogenous avian provirus ev-1 contained a defective leader segment (B. R. Cullen, A. M. Skalka, and G. Ju, Proc. Natl. Acad. Sci. USA 80:2946-2950, 1983). The sequence analysis presented here, followed by comparison with the nondefective ev-2 endogenous provirus segment, identified the critical changes at nucleotides 4 and 7 upstream of the initiator AUG. These differences do not alter the most conserved nucleotides within the consensus sequence which precedes eucaryotic initiation codons, but lie within a nine-nucleotide region that is otherwise highly conserved among avian retrovirus strains. Analysis of a series of deletion mutants indicated that other sequences within the leader are also required for efficient expression. Characterization of the altered transcripts demonstrated that the presence of the defective ev-1 segment or the deletion of a ca. 200-nucleotide leader segment did not affect the steady-state level or splicing efficiency of these mRNAs. Thus, we conclude that the reduced expression of these mRNAs is due to a translational deficiency. These results indicate that specific leader sequences, other than the previously identified consensus nucleotides which precede eucaryotic AUG initiator codons, can influence eucaryotic gene translation.

NTRE 7 is an avian retrovirus recombinant of the endogenous nononcogenic Rous-associated virus-0 (RAV-0) and the oncogenic, exogenous, transformation-defective (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 td-PrRSV-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 td-PrRSV-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.