Control of proliferation and osteogenic differentiation of human dental-pulp-derived stem cells by distinct surface structures.

The ability to control the behavior of stem cells provides crucial benefits, for example, in tissue engineering and toxicity/drug screening, which utilize the stem cell's capacity to engineer new tissues for regenerative purposes and the testing of new drugs in vitro. Recently, surface topography has been shown to influence stem cell differentiation; however, general trends are often difficult to establish due to differences in length scales, surface chemistries and detailed surface topographies. Here we apply a highly versatile screening approach to analyze the interplay of surface topographical parameters on cell attachment, morphology, proliferation and osteogenic differentiation of human mesenchymal dental-pulp-derived stem cells (DPSCs) cultured with and without osteogenic differentiation factors in the medium (ODM). Increasing the inter-pillar gap size from 1 to 6 µm for surfaces with small pillar sizes of 1 and 2 µm resulted in decreased proliferation and in more elongated cells with long pseudopodial protrusions. The same alterations of pillar topography, up to an inter-pillar gap size of 4 µm, also resulted in enhanced mineralization of DPSCs cultured without ODM, while no significant trend was observed for DPSCs cultured with ODM. Generally, cells cultured without ODM had a larger deposition of osteogenic markers on structured surfaces relative to the unstructured surfaces than what was found when culturing with ODM. We conclude that the topographical design of biomaterials can be optimized for the regulation of DPSC differentiation and speculate that the inclusion of ODM alters the ability of the cells to sense surface topographical cues. These results are essential in order to transfer the use of this highly proliferative, easily accessible stem cell into the clinic for use in cell therapy and regenerative medicine.

Spatial and temporal changes in the morphology of preosteoblastic cells seeded on microstructured tantalum surfaces.

It has been widely reported that surface morphology on the micrometer scale affects cell function as well as cell shape. In this study, we have systematically compared the influence of 13 topographically micropatterned tantalum surfaces on the temporal development of morphology, including spreading, and length of preosteoblastic cells (MC3T3-E1). Cells were examined after 0.5, 1, 4, and 24 h on different Ta microstructures with vertical dimensions (heights) of 0.25 and 1.6 mum. Cell morphologies depended upon the underlying surface topography, and the length and spreading of cells varied as a function of time with regard to the two-dimensional pattern and vertical dimension of the structure. Microstructures of parallel grooves/ridges caused elongated cell growth after 1 and 4 h in comparison to a flat, nonstructured, reference surface. For microstructures consisting of pillars, cell spreading was found to depend on the distance between the pillars with one specific pillar structure exhibiting a decreased spreading combined with a radical change in morphology of the cells. Interestingly, this morphology on the particular pillar structure was associated with a markedly different distribution of the actin cytoskeleton. Our results provide a basis for further work toward topographical guiding of cell function.

Cell volume increase in murine MC3T3-E1 pre-osteoblasts attaching onto biocompatible tantalum observed by magnetic AC mode atomic force microscopy.

Magnetic AC mode (MACmode) atomic force microscopy (AFM) was used to study murine (mouse) MC3T3-E1 preosteoblastic cells attached to biocompatible tantalum substrates. Cell volumes of attached cells derived from AFM images were compared to volumes of detached cells in suspension measured by the Coulter sizing technique. An increase of approximately 50% in cell volume was observed when the cells attached to planar tantalum substrates and developed a flattened structure including lamellipodia. We address thoroughly the issues general to the AFM determination of absolute cell volumes, and compare our magnetic AC mode AFM measurements to hitherto reported cell volume determinations by contact mode AFM.

Tumor model-specific proviral insertional mutagenesis of the Fos/Jdp2/Batf locus.

Retroviral activation of the AP-1/ATF super family member Jdp2 was recently reported to be a common event in M-MLV-induced T cell lymphoma in p27-null C57x129 mice as compared to wild type-inoculated mice but has not been found important in other models. On the basis of retroviral tag retrieval from 1190 individual Akv- and SL3-3-induced lymphomas, we here report that insertional mutagenesis into the 250-kb Fos/Jdp2/Batf locus is associated with SL3-3 MLV-induced T but not Akv-induced B cell lymphomas of NMRI and SWR mice. Integration pattern and clonality analyses suggest that Jdp2 participates in SL3-3-induced tumorigenesis distinctly as compared to the M-MLV setting. Northern blot analysis showed Jdp2 to be alternatively spliced in various normal tissues as well as MLV-induced lymphomas. Interestingly, in some tumors, proviral insertion seems to activate different mRNA sub-species. Whereas elevated mRNA levels of the Fos gene could not be correlated with provirus presence, in one case, Northern blot analysis as well as quantitative real-time PCR indicated proviral activation of the AP-1 super family member Batf, a gene not previously reported to be a target of insertional mutagenesis. A novel integration cluster between Jdp2 and Batf apparently did not influence the expression level of either gene, underscoring the importance of addressing expression effects to identify target genes of insertion. Altogether, such distinct insertion patterns point to different mechanism of activation of specific proto-oncogenes and are consequently of importance for the understanding of proviral activation mechanisms as well as the specific role of individual oncogenes in tumor development.

In vivo gene delivery to articular chondrocytes mediated by an adeno-associated virus vector.

PURPOSES: (1) To investigate the efficiency of direct in vivo adeno-associated virus (AAV) vector-mediated gene transduction to chondrocytes in relation to normal and injured articular cartilage. (2) To evaluate the effects of ultra-violet light-activated gene transduction (LAGT) in chondrocytes in vivo. (3) To determine dissemination of active rAAV vector after intra-articular administration. METHODS: Rabbit knees with either normal or injured cartilage received an intra-articular injection with 1.5x10(12) infectious rAAV-eGFP particles. The right knees received rAAV-eGFP alone, whereas the left knees were given LAGT-treatment. The transduction efficiencies were determined at 1 and 3 weeks after infection by fluorescence-activated cell scanning. The occurrence of active shedding was monitored in serum and various tissues. RESULTS: After 1 week, 7% of the chondrocytes in normal cartilage were transduced by direct rAAV transduction technique. Chondrocytes in cartilage defects demonstrated higher transduction rates compared to chondrocytes in normal cartilage. LAGT increased the cellular eGFP expression in the internal zones to 12%, but did not have any effect in the external zones in defects. Finally, infectious particles were not detected in either serum or tissue samples. CONCLUSIONS: Direct rAAV-mediated gene transfer in vivo to articular chondrocytes is possible. LAGT improves rAAV transduction of chondrocytes in vivo but appears to have a very limited range of effect induction. Expression of eGFP was not determined in other tissues than synovium and cartilage in the treated joints.

Murine leukemia virus proviral insertions between the N-ras and unr genes in B-cell lymphoma DNA affect the expression of N-ras only.

Akv1-99, a variant of Akv murine leukemia virus, induces B-cell lymphomas with nearly 100% incidence and a mean latency period of 12 months after injection into newborn NMRI mice. PCR amplification and sequence analyses of DNA flanking integrated proviruses revealed proviral insertion into the N-ras/unr (upstream of N-ras) locus in 2 out of 13 B-cell lymphomas, both of which appeared clonal by Southern blotting analysis. These two tumors showed increased expression levels of N-ras by Northern blotting, as did a third tumor shown by reverse transcriptase PCR to have a nonclonal provirus integration located in the same area. However, no significant changes in expression were observed when using a specific probe for the unr gene. All proviruses were integrated in the same transcriptional orientation as unr and N-ras genes. By promoter insertion, the two Akv1-99 proviruses integrated between exon -1 and exon 1 of N-ras gave rise to two different spliced products, whereas the provirus integrated into unr used only an exon skipping pattern. The absence of mutations of the N-ras codons 12, 13, 18, and 61 suggests that activation of the proto-oncogene is exclusively due to overexpression by retroviral promoter insertion, and furthermore, Northern blot analyses indicate that the expression of unr is unaffected by N-ras overexpression even in the case where the unr gene itself is the target of proviral insertion. Thus, altogether our findings indicate that overexpression of N-ras plays a role in development of murine leukemia virus-induced B-cell lymphomas, leaving the expression of the tightly linked unr gene unaltered.

Stromal-mediated down-regulation of CD13 in bone marrow cells originating from acute myeloid leukemia patients.

The metallopeptidase CD13 is expressed on normal myeloid cells of monocytic and granulocytic origin and on the surface of leukemic blasts in most acute myeloid leukemias (AML). To study the mechanisms regulating lineage restricted CD13 expression in AML we determined normalised CD13 mRNA levels in bone marrow cells and peripheral blood cells of 27 AML patients. Cells of bone marrow origin had lower levels of normalised CD13 mRNA than cells of peripheral blood origin, even though fluorescence intensity and fraction of cells expressing CD13 on the surface was unchanged. In particular, AML patients with very low levels of normalised CD13 mRNA in bone marrow cells showed an increase in CD13 mRNA expression in peripheral blood. To evaluate the effects of bone marrow microenvironment on CD13 mRNA expression, we cultured leukemic myeloid cells with and without murine stromal cells. Bone marrow cells with high and low CD13 surface expression that entered the stromal layers all down-regulated CD13 mRNA expression as compared to cells in suspension above. For peripheral blood cells within stromal layers, CD13 mRNA expression was diminished in only 3 out of 6 cases. The ambiguous effect of stromal cells on peripheral blood cells may illustrate a differentiation-dependent response towards stroma. We determined the polyadenylation status of CD13 mRNA for 9 bone marrow aspirates and 7 peripheral blood samples. Polyadenylation was diminished in bone marrow cells from AML patients with low levels of normalised CD13 mRNA, raising the possibility of involvement of mRNA instability in regulation of CD13 mRNA expression in this subgroup of patients.

Transfer of primer binding site-mutated simian immunodeficiency virus vectors by genetically engineered artificial and hybrid tRNA-like primers.

Simian immunodeficiency viruses (SIV) harbor primer binding sites (PBS) matching tRNA or tRNA. To study determinants of primer usage in SIV, a SIVmac239-based vector was impaired by mutating the PBS to a sequence (PBS-X2) with no match to any tRNA. By cotransfection of a synthetic gene encoding a tRNA(Pro)-like RNA with a match to PBS-X2, the activity of this vector could be restored to a transduction efficiency slightly lower than that of the wild-type vector. A vector with a PBS matching tRNA(Pro) was functional at a level slightly below that of the wild-type vector, but higher transduction efficiency could be obtained by cotransfection of a gene for an engineered tRNA(Pro)-tRNA hybrid with a match to PBS-Pro. The importance of tRNA backbone identity was further analyzed by complementing the PBS-X2 vector with a gene for a matching x2 primer with a tRNA backbone, which led to three- to fourfold-higher titers than those observed for the x2 primer with the tRNA(Pro) backbone. In summary, our results demonstrate flexibility in PBS and primer usage for SIVmac239, with PBS-primer complementarity being the major determinant, in analogy with previous findings for murine leukemia viruses and human immunodeficiency virus type 1.

Single site polymorphisms and alternative splicing of the human CD13 gene--different splicing frequencies among patients with acute myeloid leukaemia and healthy individuals.

Within the haematopoietic system, CD13/aminopeptidase N (APN), a transmembrane glycoprotein, is expressed on the surface of early committed progenitors of granulocytes and monocytes and by all cells of these lineages as they mature. CD13 is expressed on the majority of leukaemic myeloblasts in acute myeloid leukaemia (AML), and on leukaemic lymphoblasts in a small percentage of acute lymphoid leukaemia cases. Thus, anti-CD13 monoclonal antibodies are used as diagnostic markers in leukaemia typing. By systematically amplifying overlapping reverse transcription polymerase chain reaction (RT-PCR) amplicons throughout the CD13 mRNA, we identified two splice variants in which exon 3 and exon 14 were lost. Fourteen healthy individuals and 34 patients with AML were screened for these splice variants. All healthy individuals, and the majority of AML patients, had both splice variants but they represented less than 10% of the total RT-PCR-amplified CD13 product. Increased expression of both truncated CD13 mRNA forms were observed in 6% of AML patients, whereas no detectable exon 3 or exon 14 splice variants could be generated in 26% and 9% of AML patients respectively. The different splicing frequencies may reflect altered processing of pre-mRNA or expansion of certain cell types for some AML patients, even though no correlation existed to blast percentage, FAB classification, surface antigens or cytogenetic characteristics. In addition, we identified an intron of 506 bp between exon 1 and exon 2 as well as two sites of single nucleotide polymorphism with a heterozygosity index of about 0.5, making them useful as genetic markers.

Expression of type III sodium-dependent phosphate transporters/retroviral receptors mRNAs during osteoblast differentiation.

Inorganic phosphate (Pi) is essential for the formation of bone. Pi transport in osteoblastic cells is mainly handled by sodium-dependent Pi (NaPi) transporters, different from the renal type I and II transporters; their molecular identities are, however, still subject to investigation. Recently, two type III NaPi transporters, Pit1 and Pit2, were identified, both of which exhibit some of the biochemical and regulatory characteristics of osteoblastic cell-associated NaPi transporters. Here, we have investigated the Pit1 and Pit2 steady-state mRNA levels during the osteoblast differentiation in cultures of the nontransformed MC3T3-E1 cell line. While Pit2 mRNAs were invariably expressed at low levels, Pit1 mRNA levels were found to increase during osteoblast differentiation concomitantly with osteocalcin mRNA. Moreover, the increase in Pit1 mRNA levels also correlated with the time in culture at which mineralization could be observed. The increase in Pit1 mRNA levels over time in culture was only observed in cultures grown under conditions allowing for osteoblast differentiation. This is the first time that osteoblast differentiation-dependent regulation of expression of a NaPi transporter has been demonstrated. Moreover, we show here for the first time the presence of Pit1 and Pit2 mRNAs in undifferentiated and differentiated nontransformed osteoblastic cells. Our data suggest that both Pit1 and Pit2 NaPi transporters are involved in Pi transport in preosteoblastic and osteoblastic cells, and they represent the first evidence consistent with a potential role for Pit1, but not for Pit2, in differentiation-dependent Pi transport. The observed upregulation of Pit1 mRNA levels during osteoblast differentiation suggests that Pit1 might be used as a marker for osteoblast maturation.

Functional screening of a retroviral peptide library for MHC class I presentation.

We have used retroviral vector technology to develop a method for functional screening of combinatorial peptide libraries expressed inside mammalian cells with the ultimate goal of identifying new drug targets. The method was validated in a library screening experiment based on antigen presentation of small peptides. A library encoding SIXNXEKX-peptides, where X designates randomised positions corresponding to major histocompatibility (MHC) class I anchor residues, was generated in a retroviral vector. The library was transduced into a population of antigen presenting cells (APCs) known to mediate MHC class I restricted presentation of the SIINFEKL peptide. The cellular library was screened by using an antigen presentation assay in which a T cell hybridoma recognising the MHC class I/SIINFEKL peptide complex was employed. Using this experimental model, we identified two positive cellular clones both encoding SIINFEKL peptides with identical codon usage. This number corresponded well to the expected frequency of SIINFEKL in the library. The lack of identification of other peptides capable of activating the T-hybridoma supports previous findings of a high degree of specificity at the level of peptide-loading of MHC-molecules. The result further demonstrates the potential of using combinatorial libraries for functional screening and selection of effector peptides stably expressed in mammalian cells.

Alleviation of murine leukemia virus repression in embryonic carcinoma cells by genetically engineered primer binding sites and artificial tRNA primers.

The primer binding site (PBS) plays pivotal roles during reverse transcription of retroviruses and also is the target of a cellular host defense impeding the transcription of murine leukemia virus (MLV) harboring a proline (pro) PBS in embryonic cells. Both the PBS and the tRNA primer are copied during reverse transcription and anneal as complementary DNA sequences creating the PBS of the integrated provirus. The pro PBS of MLV can be exchanged by PBS sequences matching endogenous or engineered tRNAs to allow replication of Akv MLV-derived vectors in fibroblasts. Here we use the PBS escape mutant B2 to demonstrate the capacity of the synthetic tRNA(B2) to function in reverse transcription in competition with endogenous tRNAs in fibroblasts and embryonic carcinoma (EC) cells. We further show symmetry between PBS and the primer by the ability of the synthetic tRNA(B2) to confer escape from EC repression of a PBS-Pro vector. Of a panel of vectors with the repressed pro PBS substituted for other natural or artificial PBS sequences, all except one efficiently expressed the neo marker gene when transferred to NIH/3T3 and EC cells, hence avoiding PBS-mediated silencing in EC cells. A non-natural PBS matching an artificially designed tRNA molecule conferred no further relief from repression than that attained with the B2 escape mutant or the natural alternative PBSs. Interestingly, a vector harboring a PBS matching tRNA(Lys1.2) suffered repression similar to the wild-type PBS-Pro but was partially rescued by a single point mutation of the PBS.

Lack of shielding of primer binding site silencer-mediated repression of an internal promoter in a retrovirus vector by the putative insulators scs, BEAD-1, and HS4.

A major determinant for transcriptional incompetence of murine leukemia virus (MLV) and MLV-derived vectors in embryonal cells is located at the proline primer binding site (PBS). The mechanism of silencing is unknown, yet the effect is capable of spreading to adjacent promoters. Based on a retroviral vector containing an internal promoter and the escape mutant B2 PBS with expressional capacity in embryonal cells, we have developed an assay to test the ability of putative insulators to shield the silencer at the PBS. Since the B2 PBS reverts to the wild-type PBS at high frequency, a shielding ability of a putative insulator can be assessed from the ratio of expressing B2 PBS to proline PBS proviruses in the target embryonal carcinoma cell population as measured by primer extension. Our results show that none of the possible insulators, scs, BEAD-1, or HS4, is able to shield an internal promoter from the repressive effect of the silencer at the PBS region when inserted between the silencer and the promoter.

Identification of a novel human tRNA(Ser(CGA)) functional in murine leukemia virus replication.

We have identified a human tRNA(Ser) isoacceptor matching the UCG codon. The tRNA was discovered via its ability to act in reverse transcription of a murine leukemia virus vector containing a complementary tRNA primer binding site (Lund et al., Nucleic Acids Res., 28 (2000) 791-799). The tRNA(Ser(CGA)) was detected in cell lines of human, monkey and mouse origin. The UCG codon is the most rarely used codon in human genes. The cloned human tRNA(Ser(CGA)) gene encodes an 85 nucleotide, intron-less tRNA, contains a consensus split intragenic promoter and is located at region p21.3-22.2 on chromosome 6. The integrity and functionality of the cloned tRNA(Ser(CGA)) gene was verified by in vitro transcription analysis in HeLa nuclear extracts.

Genetic reassortment and patch repair by recombination in retroviruses.

Retroviral particles contain a diploid RNA genome which serves as template for the synthesis of double-stranded DNA in a complex process guided by virus-encoded reverse transcriptase. The dimeric nature of the genome allows the proceeding polymerase to switch templates during copying of the copackaged RNA molecules, leading to the generation of recombinant proviruses that harbor genetic information derived from both parental RNAs. Template switching abilities of reverse transcriptase facilitate the development of mosaic retroviruses with altered functional properties and thereby contribute to the restoration and evolution of retroviruses facing altering selective forces of their environment. This review focuses on the genetic patchwork of retroviruses and how mixing of sequence patches by recombination may lead to repair in terms of re-established replication and facilitate increased viral fitness, enhanced pathogenic potential, and altered virus tropisms. Endogenous retroelements represent an affluent source of functional viral sequences which may hitchhike with virions and serve as sequence donors in patch repair. We describe here the involvement of endogenous viruses in genetic reassortment and patch repair and review important examples derived from cell culture and animal studies. Moreover, we discuss how the patch repair phenomenon may challenge both safe usage of retrovirus-based gene vehicles in human gene therapy and the use of animal organs as xenografts in humans. Finally, the ongoing mixing of distinct human immunodeficiency virus strains and its implications for antiviral treatment is discussed.

Sint1, a common integration site in SL3-3-induced T-cell lymphomas, harbors a putative proto-oncogene with homology to the septin gene family.

The murine retrovirus SL3-3 is a potent inducer of T-cell lymphomas when inoculated into susceptible newborn mice. Previously, DNAs from twenty SL3-3-induced tumors were screened by PCR for provirus integration sites. Two out of 20 tumors demonstrated clonal provirus insertion into a common region. This region has now been isolated and characterized. The region, named SL3-3 integration site 1 (Sint1), maps to the distal end of mouse chromosome 11, corresponding to human chromosome 17q25, and may be identical to a mouse mammary tumor virus integration site in a T-cell lymphoma, Pad3. Two overlapping genomic lambda clones spanning about 35 kb were isolated and used as a starting point for a search for genes in the neighborhood of the virus integration sites. A genomic fragment was used as a hybridization probe to isolate a 3-kb cDNA clone, the expression of which was upregulated in one of two tumors harboring a provirus in Sint1. The cDNA clone is predicted to encode a protein which shows 97.0% identity to a human septin-like protein encoded by a gene which has been found as a fusion partner gene of MLL in an acute myeloid leukemia with a t(11;17)(q23;q25). Together these findings raise the possibility that a proto-oncogene belonging to the septin family, and located about 15 kb upstream of the provirus integration sites, is involved in murine leukemia virus-induced T-cell lymphomagenesis.

A 13-amino-acid Pit1-specific loop 4 sequence confers feline leukemia virus subgroup B receptor function upon Pit2.

Feline leukemia virus subgroup B (FeLV-B) and gibbon ape leukemia virus (GALV) utilize the human protein Pit1 but not the related protein, Pit2, as receptor. A stretch of 9 amino acids, named region A, was identified in the putative fourth extracellular loop of Pit1 (residues 550 through 558) as critical for FeLV-B and GALV receptor function. However, the presence of Pit1 region A did not confer receptor function for FeLV-B upon Pit2, while it did so for GALV. We have here shown that the presence of two Pit1-specific loop 4 residues (tyrosine 546 and valine 548) in addition to Pit1 region A is sufficient to make Pit2 an efficient FeLV-B receptor; that is, a stretch of 13 amino acids encompassing all loop 4 amino acid differences between Pit1 and Pit2 comprises a C-terminal determinant for FeLV-B receptor function. Thus, the same limited receptor region is sufficient to confer receptor function for both viruses upon Pit2.

Mutations of the kissing-loop dimerization sequence influence the site specificity of murine leukemia virus recombination in vivo.

The genetic information of retroviruses is retained within a dimeric RNA genome held together by intermolecular RNA-RNA interactions near the 5' ends. Coencapsidation of retrovirus-derived RNA molecules allows frequent template switching of the virus-encoded reverse transcriptase during DNA synthesis in newly infected cells. We have previously shown that template shifts within the 5' leader of murine leukemia viruses occur preferentially within the kissing stem-loop motif, a cis element crucial for in vitro RNA dimer formation. By use of a forced recombination approach based on single-cycle transfer of Akv murine leukemia virus-based vectors harboring defective primer binding site sequences, we now report that modifications of the kissing-loop structure, ranging from a deletion of the entire sequence to introduction of a single point mutation in the loop motif, significantly disturb site specificity of recombination within the highly structured 5' leader region. In addition, we find that an intact kissing-loop sequence favors optimal RNA encapsidation and vector transduction. Our data are consistent with the kissing-loop dimerization model and suggest that a direct intermolecular RNA-RNA interaction, here mediated by palindromic loop sequences within the mature genomic RNA dimer, facilitates hotspot template switching during retroviral cDNA synthesis in vivo.

Selection of functional tRNA primers and primer binding site sequences from a retroviral combinatorial library: identification of new functional tRNA primers in murine leukemia virus replication.

Retroviral reverse transcription is initiated from a cellular tRNA molecule and all known exogenous isolates of murine leukemia virus utilise a tRNA(Pro)molecule. While several studies suggest flexibility in murine leukemia virus primer utilisation, studies on human immunodeficiency virus and avian retro-viruses have revealed evidence of molecular adapt-ation towards the specific tRNA isoacceptor used as replication primer. In this study, murine leukemia virus tRNA utilisation is investigated by in vivo screening of a retroviral vector combinatorial library with randomised primer binding sites. While most of the selected primer binding sites are complementary to the 3'-end of tRNA((Pro)), we also retrieved PBS sequences matching four other tRNA molecules and demonstrate that Akv murine leukemia virus vectors may efficiently replicate using tRNA(Arg(CCU)), tRNA(Phe(GAA))and a hitherto unknown human tRNA(Ser(CGA)).