Reduced Mcm2 expression results in severe stem/progenitor cell deficiency and cancer.

Mcm2 is a component of the DNA replication licensing complex that marks DNA replication origins during G1 of the cell cycle for use in the subsequent S-phase. It is expressed in stem/progenitor cells in a variety of regenerative tissues in mammals. Here, we have used the Mcm2 gene to develop a transgenic mouse in which somatic stem/progenitor cells can be genetically modified in the adult. In these mice, a tamoxifen-inducible form of Cre recombinase is integrated 3' to the Mcm2 coding sequence and expressed via an internal ribosome entry site (IRES). Heterozygous Mcm2(IRES-CreERT2/wild-type (wt)) mice are phenotypically indistinguishable from wild-type at least through 1 year of age. In bigenic Mcm2(IRES-CreERT2/wt); Z/EG reporter mice, tamoxifen-dependent enhanced green fluorescence protein expression is inducible in a wide variety of somatic stem cells and their progeny. However, in Mcm2(IRES-CreERT2/IRES-CreERT2) homozygous embryos or mouse embryonic fibroblasts, Mcm2 is reduced to approximately one-third of wild-type levels. Despite the fact that these mice develop normally and are asymptomatic as young adults, life span is greatly reduced, with most surviving to only approximately 10-12 weeks of age. They demonstrate severe deficiencies in the proliferative cell compartments of a variety of tissues, including the subventricular zone of the brain, muscle, and intestinal crypts. However, the immediate cause of death in most of these animals is cancer, where the majority develop lymphomas. These studies directly demonstrate that deficiencies in the function of the core DNA replication machinery that are compatible with development and survival nonetheless result in a chronic phenotype leading to stem cell deficiency in multiple tissues and cancer. Disclosure of potential conflicts of interest is found at the end of this article.

Stem/progenitor cell-specific enhanced green fluorescent protein expression driven by the endogenous Mcm2 promoter.

Previous studies have demonstrated expression of the minichromosome maintenance protein Mcm2 in cells that remain competent to divide, including stem/progenitor cells of the subventricular zone (SVZ) within the brain. Here, a transgenic mouse line in which the Mcm2 gene drives expression of enhanced green fluorescent protein (EGFP) was constructed by insertion of an internal ribosomal entry site (IRES)-EGFP cassette into the last exon of the gene, 3' to the stop codon. In these mice, expression of EGFP is observed in the SVZ and several other tissues with high proliferative activity, including the spleen, intestine, hair follicles, and bone marrow. These observations suggest that EGFP fluorescence in this mouse line provides an index of the proliferative capacity of different tissues. Immunohistological analysis demonstrates a direct concordance between expression of EGFP and Mcm2, consistent with a transcriptional level downregulation of Mcm2 expression in postmitotic cells. To test the utility of EGFP expression for recovery of live cells retaining the capacity to divide, EGFP-expressing and -nonexpressing cells from bone marrow and brain were isolated from an adult Mcm2(IRES-EGFP) mouse by fluorescence-activated cell sorting and assayed for clonal growth. The EGFP-positive fraction contained the entire clonogenic population of the bone marrow and greater than 90% of neurosphere-forming cells from the brain. Brain-derived clonogenic cells were shown to remain competent to differentiate towards all three neural lineages. These studies demonstrate that the Mcm2(IRES-EGFP) transgenic line constructed here can be used for recovery of proliferation competent cells from different tissue types.

Accumulation of mutations and somatic selection in aging neural stem/progenitor cells.

Genomic instability within somatic stem cells may lead to the accumulation of mutations and contribute to cancer or other age-related phenotypes. However, determining the frequency of mutations that differ among individual stem cells is difficult from whole tissue samples because each event is diluted in the total population of both stem cells and differentiated tissue. Here the ability to expand neural stem/progenitor cells clonally permitted measurement of genomic alterations derived from a single initial cell. C57Bl/6 x DBA/2 hybrid mice were used and PCR analysis with strain-specific primers was performed to detect loss of heterozygosity on nine different chromosomes for each neurosphere. The frequency with which changes occurred in neurospheres derived from 2-month- and 2-year-old mice was compared. In 15 neurospheres derived from young animals both parental chromosomes were present for all nine chromosome pairs. In contrast, 16/17 neurospheres from old animals demonstrated loss of heterozygosity (LOH) on one or more chromosomes and seven exhibited a complete deletion of at least one chromosomal region. For chromosomes 9 and 19 there is a significant bias in the allele that is lost where in each case the C57Bl/6 allele is retained in 6/6 neurospheres exhibiting LOH. These data suggest that aging leads to a substantial mutational load within the neural stem cell compartment which can be expected to affect the normal function of these cells. Furthermore, the retention of specific alleles for chromosomes 9 and 19 suggests that a subset of mutational events lead to an allele-specific survival advantage within the neural stem cell compartment.