Molecular Signature of Migratory Human Mesenchymal Stromal Cells; Influence of the Cell Cycle.

Abstract 1450

Poster Board I-473

Mesenchymal stromal cells (MSC) are a potential cell source for cellular therapies, in which recruitment and migration of MSC towards injured tissue is crucial. However, in vitro and in vivo experiments reveal that the capacity to migrate and home to sites of injury is limited and huge cell numbers have to be transplanted. Therefore, better understanding of the mechanisms of MSC migration will improve the design and efficacy of future cellular therapies. With respect to these therapies, we are studying the process of migration in MSC.

In Transwell migration experiments it was observed that MSC derived from various tissues all contain only a small percentage (10-25%) of migratory MSC, which was determined to be the same cell fraction migrating towards various chemokines. This migratory fraction could not be defined as a specific subpopulation by surface marker expression. Interestingly, actin rearrangement and increased paxillin phosphorylation were observed in the majority of the MSC upon stimulation with chemokines. This indicates that functionality of the machinery involved in the initial response to migratory cues is not restricted to the migratory MSC subset. However, the migratory MSC fraction contained significantly less cells in S- and G2/M-phase (ratio S:0.81±0.13, p<0.028; G2/M: 0.75±0.13, p<0.031) as compared to non-migrating MSC. Ki67 antigen expression, which discriminates between G0- and G1-phase, revealed a trend of more cells in G1-phase in migratory MSC. A similar role for the cell cycle in homing and mobilization of hematopoietic stem cells has been described previously (1). Here we report for the first time that the cell cycle also affects MSC migration.

To further study the molecular signature of the migratory MSC, a micro array was performed on migrating and non-migrating fetal bone marrow MSC, SDF-1 was used as chemokine. MSC that were only exposed to a SDF-1 gradient and cultured fetal bone marrow MSC were included as controls. SDF-1 exposure induced differential expression of 674 genes (383 up, 291 down) compared to cultured MSC. This list is enriched for genes involved in the (regulation of) cell cycle, response to wound healing and regulation of cell differentiation. These results indicate that besides promoting MSC migration, SDF-1 also induces other (paracrine) functions that MSC may have in the injured niche. A remarkable small number of genes was differentially expressed between migrating and non-migrating MSC. Nurr1, Nur77, CYR61, SMAD7, AXIN1 and ID3 were upregulated (range 1,5 to 2.3-fold), HIST1H2AK and HIST1H4B were downregulated (-1.5 fold). These results were confirmed by RQ-PCR. Only for the two genes of the nuclear orphan receptor family (Nurr1 and Nur77) the upregulation was >2-fold. Therefore these genes were studied in more detail. After 4 hrs exposure to SDF-1, expression levels of Nurr1 and Nur77 were increased 14 and 5 fold respectively in fetal bone marrow-derived MSC as compared to MSC that were not exposed to SDF-1. Another chemoattractant for MSC (PDGF-BB) also induced their expression, but less pronounced. Nurr1 and Nur77 are members of the nuclear orphan receptor family and were first described as early response transcription factors upon growth factor stimulation (2). More recently, roles in the inflammatory response (downregulation of cytokines) have been described (3). The functional requirement of Nurr1 and Nur77 in MSC was studied by lentiviral knock down of Nur77 or Nurr1. This resulted in a 3-4 fold increase of MSC in S- and G2/M phase of the cell cycle compared to the scrambled control and is in agreement with the observation that overexpression of Nur77 inhibits the cell cycle by inducing P27^kip (3). From these knock down studies, micro array results and our observation that S and G2/M phase negatively influence MSC migration, it could be hypothesized that overexpression of Nur77 and Nurr1 will lead to increased MSC migration. In conclusion, our results have identified a role for cell cycle in MSC migration. Similar to HSC, S- and G2/M-phase negatively influence MSC migration. Considering the possible functions in MSC homing and in modulating the immune response, unraveling the role of Nur77 and Nurr1 in MSC may have dual implications for future regenerative medicine.