Human bone marrow mesenchymal stem cells (hMSCs) represent an appealing source of adult stem cells for cell therapy and tissue engineering, as they are easily obtained and expanded while maintaining their multilineage differentiation potential. All current protocols for in vitro culture of hMSCs include fetal bovine serum (FBS) as nutritional supplement. FBS is an undesirable additive to cells that are expanded for therapeutic purposes in humans because the use of FBS carries the risk of transmitting viral and prion diseases and proteins that may initiate xenogeneic immune responses. In the present study, we have therefore investigated if autologous serum (AS) or allogeneic human serum (alloHS) could replace FBS for the expansion of hMSCs in vitro. We discovered that the choice of serum affected hMSCs at several different levels.First, hMSCs in AS proliferated markedly faster than hMSCs in FBS, whereas use of alloHS resulted in hMSC growth arrest and death. Second, hMSCs in FBS differentiated more rapidly toward mesenchymal lineages compared with hMSCs in AS. Interestingly, genome-wide microarray analysis identified several transcripts involved in cell cycle and differentiation that were differentially regulated between hMSCs in FBS and AS. Finally, several transcripts, including some involved in cell cycle inhibition, were upregulated in hMSCs in FBS at a late passage, whereas the hMSC transcriptome in AS was remarkably stable. Thus, hMSCs may be expanded rapidly and with stable gene expression in AS in the absence of growth factors, whereas FBS induces a more differentiated and less stable transcriptional profile. Stem Cells 2005;23:1357-1366

Base excision repair (BER) is initiated by a DNA glycosylase and is completed by alternative routes, one of which requires proliferating cell nuclear antigen (PCNA) and other proteins also involved in DNA replication. We report that the major nuclear uracil-DNA glycosylase (UNG2) increases in S phase, during which it co-localizes with incorporated BrdUrd in replication foci. Uracil is rapidly removed from replicatively incorporated dUMP residues in isolated nuclei. Neutralizing antibodies to UNG2 inhibit this removal, indicating that UNG2 is the major uracil-DNA glycosylase responsible. PCNA and replication protein A (RPA) co-localize with UNG2 in replication foci, and a direct molecular interaction of UNG2 with PCNA (one binding site) and RPA (two binding sites) was demonstrated using two-hybrid assays, a peptide SPOT assay and enzyme-linked immunosorbent assays. These results demonstrate rapid post-replicative removal of incorporated uracil by UNG2 and indicate the formation of a BER complex that contains UNG2, RPA and PCNA close to the replication fork.

We have expressed a human recombinant uracil-DNA glycosylase (UNG delta 84) closely resembling the mature form of the human enzyme (UNG, from the UNG gene) in Escherichia coli and purified the protein to apparent homogeneity. This form, which lacks the first seven nonconserved amino acids at the amino terminus, has properties similar to a 50% homogeneous UDG purified from human placenta except for a lower salt optimum and a slightly lower specific activity. The recombinant enzyme removed U from ssDNA approximately 3-fold more rapidly than from dsDNA. In the presence of 10 mM NaCl, Km values were 0.45 and 1.6 microM with ssDNA and dsDNA, respectively, but Km values increased significantly with higher NaCl concentrations. The pH optimum for UNG delta 84 was 7.7-8.0; the activation energy, 50.6 kJ/mol; and the pI between 10.4 and 10.8. The enzyme displays a striking sequence specificity in removal of U from UA base pairs in M13 dsDNA. The sequence specificity for removal of U from UG mismatches (simulating the situation after deamination of C) was essentially similar to removal from UA matches when examined in oligonucleotides. However, removal of U from UG mismatches was in general slightly faster, and in some cases significantly faster, than removal from UA base pairs. Immunofluorescence studies using polyclonal antibodies against UNG delta 84 demonstrated that the major fraction of UNG was located in the nucleus. Furthermore, > 98% of the total uracil-DNA glycosylase activity from HeLa cell extracts was inhibited by the antibodies, indicating that the UNG protein represents the major uracil-DNA glycosylase in the cells.

A distinct nuclear form of human uracil-DNA glycosylase [UNG2, open reading frame (ORF) 313 amino acid residues] from the UNG gene has been identified. UNG2 differs from the previously known form (UNG1, ORF 304 amino acid residues) in the 44 amino acids of the N-terminal sequence, which is not necessary for catalytic activity. The rest of the sequence and the catalytic domain, altogether 269 amino acids, are identical. The alternative N-terminal sequence in UNG2 arises by splicing of a previously unrecognized exon (exon 1A) into a consensus splice site after codon 35 in exon 1B (previously designated exon 1). The UNG1 sequence starts at codon 1 in exon 1B and thus has 35 amino acids not present in UNG2. Coupled transcription/translation in rabbit reticulocyte lysates demonstrated that both proteins are catalytically active. Similar forms of UNG1 and UNG2 are expressed in mouse which has an identical organization of the homologous gene. Constructs that express fusion products of UNG1 or UNG2 and green fluorescent protein (EGFP) were used to study the significance of the N-terminal sequences in UNG1 and UNG2 for subcellular targeting. After transient transfection of HeLa cells, the pUNG1-EGFP-N1 product colocalizes with mitochondria, whereas the pUNG2-EGFP-N1 product is targeted exclusively to nuclei.