Atmospheric oxygen inhibits growth and differentiation of marrow-derived mouse mesenchymal stem cells via a p53-dependent mechanism: implications for long-term culture expansion.

Large scale expansion of human mesenchymal stem cells (MSCs) is routinely performed for clinical therapy. In contrast, developing protocols for large scale expansion of primary mouse MSCs has been more difficult due to unique aspects of rodent biology. Currently, established methods to isolate mouse MSCs select for rapidly dividing subpopulations that emerge from bone marrow cultures following long-term (months) expansion in atmospheric oxygen. Herein, we demonstrate that exposure to atmospheric oxygen rapidly induced p53, TOP2A, and BCL2-associated X protein (BAX) expression and mitochondrial reactive oxygen species (ROS) generation in primary mouse MSCs resulting in oxidative stress, reduced cell viability, and inhibition of cell proliferation. Alternatively, procurement and culture in 5% oxygen supported more prolific expansion of the CD45(-ve) /CD44(+ve) cell fraction in marrow, produced increased MSC yields following immunodepletion, and supported sustained MSC growth resulting in a 2,300-fold increase in cumulative cell yield by fourth passage. MSCs cultured in 5% oxygen also exhibited enhanced trilineage differentiation. The oxygen-induced stress response was dependent upon p53 since siRNA-mediated knockdown of p53 in wild-type cells or exposure of p53(-/-) MSCs to atmospheric oxygen failed to induce ROS generation, reduce viability, or arrest cell growth. These data indicate that long-term culture expansion of mouse MSCs in atmospheric oxygen selects for clones with absent or impaired p53 function, which allows cells to escape oxygen-induced growth inhibition. In contrast, expansion in 5% oxygen generates large numbers of primary mouse MSCs that retain sensitivity to atmospheric oxygen, and therefore a functional p53 protein, even after long-term expansion in vitro.

Fibroblast growth factor 2 (Fgf2) inhibits differentiation of mesenchymal stem cells by inducing Twist2 and Spry4, blocking extracellular regulated kinase activation, and altering Fgf receptor expression levels.

Mesenchymal stem cells (MSCs) are known to differentiate into connective tissue lineages but intracellular signaling pathways that maintain cells in an undifferentiated state remain largely unexplored. Previously, we reported that fibroblast growth factor 2 (Fgf2) reversibly inhibited multilineage differentiation of primary mouse MSCs and now identify a unique compliment of signaling proteins that are dynamically regulated by this mitogen and whose expression levels are strongly correlated with inhibition of cell differentiation. Fgf2 selectively induced expression of Twist2 and Sprouty4 (Spry4) and repressed expression of soluble frizzled related receptor 2 (Sfrp2), runt-related transcription factor 2 (Runx2), and peroxisome proliferation activated receptor gamma (Pparg). In contrast, Wnt3a induced expression of Twist but not Twist2 or Spry4 and bone morphogenetic protein 2 (Bmp2) failed to alter expression of all three genes. Moreover, pretreatment of MSCs with Fgf2 delayed extracellular regulated kinase 1 (Erk1) and Erk2 phosphorylation and repressed bone-specific gene expression during an osteoinduction time course. Alternatively, pretreatment with Wnt3a had no effect, whereas Bmp2 pretreatment augmented Erk1/2 activation and bone-specific gene expression. Fgf2 also induced expression of Fgf receptor 1 (Fgfr1) and Fgfr4 and repressed Fgfr2 and Fgfr3 expression in MSCs, whereas Wnt3a and Bmp2 had the opposite effect. Finally, immunostaining revealed that Twist and Spry4 were coexpressed in MSCs and that Fgf2 treatment altered their subcellular distribution in a manner consistent with their mode of action. Collectively, these studies demonstrate that inhibition of mouse MSC differentiation by Fgf2 is strongly correlated with upregulation of Twist2 and Spry4 and suppression of Erk1/2 activation.

Murine mesenchymal stem cells transplanted to the central nervous system of neonatal versus adult mice exhibit distinct engraftment kinetics and express receptors that guide neuronal cell migration.

Mesenchymal stem cells (MSCs) have demonstrated efficacy as cellular vectors for treating a variety of nervous system disorders. Nevertheless, few studies have quantified MSC engraftment levels or explored the mechanisms that promote their survival and migration in nervous tissue. In this study, we compared the engraftment kinetics and anatomical distribution of murine, male MSCs injected intracranially into neonatal versus adult female mice using a real-time PCR assay that targets the mouse SRY gene. These analyses revealed that MSCs exhibited low but equivalent engraftment levels in the central nervous system (CNS) of neonatal and adult transplant recipients at 12 days post-injection. However, MSC engraftment levels were significantly greater at 60 and 150 days post-transplantation in neonates as compared to adults. Despite these differences, engrafted MSCs were widely distributed along the neuraxis of the CNS in both transplant groups. Collectively, these data indicate that proliferation, but not engraftment and migration, of MSCs in brain are regulated by the host microenvironment. Using a genomics approach, we also identified MSC subpopulations that express neural adhesion proteins and receptors that regulate neuronal cell migration in brain, including cadherin 2, neurexin 1, ninjurin 1, neogenin 1, neuropilin 2, and roundabout homolog 1 and 4. Functional studies indicate these proteins confer cell adhesion and migration of MSCs in response to the appropriate chemoattractant. On the basis of these findings, we conclude that the unique molecular composition of MSC subpopulations imparts to them an inherent capacity to engraft and migrate in brain. These subpopulations may represent more potent cellular vectors for treating CNS disorders.

The structure of the Escherichia coli O148 lipopolysaccharide core region and its linkage to the O-specific polysaccharide.

Recently it was demonstrated that Shigella dysenteriae type 1, a cause of severe dysentery epidemics, gained its O-specific polysaccharide (O-SP) from Escherichia coli O148. The O-SPs of these bacteria differ only by a galactose residue in the repeat unit of S. dysenteriae type 1 in place of a glucose residue in E. coli O148. Herein, we analyzed the core structure and its linkage to the O-SP in E. coli O148 LPS. Both were found to be identical to those of S. dysenteriae type 1 structures, further supporting the relatedness of these two bacteria. The following structure of the core with one repeat unit of the O-SP has been assigned (all have d-configuration except l-Rha):