Answer to referee 1 comment:
1. Mobilized peripheral blood is a more clinical source of CD34+ cells, so it is necessary to compare the MMP-9 secretion and trans-migration ability of CB CD34+ cells with that of mobilized PB CD34+ cells. However, we couldn't obtain enough mobilized PB to separate PB CD34+ cells and determine the MMP-9 secretion and migration ability, so we couldn’t complement the study on PB CD34+ cells in this paper. Results obtained by Janowska-Wieczorek et al found that mobilized CD34+ cells in peripheral blood express MMP-9. Furthermore, Domenech’s study showed that MMP-9 secretion is involved in G-CSF induced HPC mobilization. Their conclusions have been added in the discussion. In our present study, our central conclusion from our data is that freshly isolated CD34+ stem/progenitor cells obtained from CB produce MMP-9.
2. MMP-9 negative cell used in fig 1C was Jurkat cell. In zymographic analysis, MMP-9 was not detected in the medium conditioned by Jurkat cell. To exclude that the contaminating cells may play a role in the observed MMP-9 production, we screened the media conditioned by different proportion of CB mononuclear cells with MMP-9 negative cells by zymography. This result may be confusion. Actually, only by detecting the medium conditioned by 2X105 CB mononuclear cells (MNC)/ml (since the purities of CD34+ cell are more than 90%), it could exclude the MNC role. In the revised manuscript, we only detected MMP-9 activity and antigen level in the medium conditioned by 2X105 CB mononuclear cells (MNC)/ml. There is no MMP-9 secretion be detected in the medium conditioned by 2X105 CB MNC/ml. It excluded the possibility that the MMP-9 activity in CB CD34+ cells conditioned medium is due to the contamination by MNC.
3.In this revised paper, we have detected the MMP-9 antigen levels by using commercial specific ELISA kits (R&D System, sensitivity, 0.156ng/ml). Recombinant MMP-9 from R&D System was used as a standard. The results are expressed in the absolute concentration. The absolute concentration result has been added in the paper. As shown in Fig2, MMP-9 levels were detectable in both CB CD34+ cell conditioned medium and BM CD34+ cell conditioned medium. However, MMP-9 level was significantly higher in CB CD34+ cell conditioned medium than in BM CD34+ cell conditioned medium (0.406±0.133ng/ml versus 0.195±0.023ng/ml). Although gelatinolytic activity was not detected in media conditioned by CD34+ cells from BM, sensitivity of ELISA favors the detection of MMP-9 antigen in the BM CD34+.
4. In our study, to establish the direct link between MMP-9 and CB CD34+ cells migration, we only determined the role of MMP-9 in spontaneous migration of CB CD34+ cells, but not in chemotactic migration. Actually, regulation of hematopoietic stem cell migration, homing and anchorage of repopulation cells to the bone marrow involves a complex interplay between adhesion molecules, chemokines, cytokines and proteolytic enzymes. Results obtained by the groups of Voermans reveal that not only the spontaneous migration but also the SDF-1 induced migration of CB CD34+ cells is greatly increased in comparison to CD34+ cells from BM and peripheral blood.
5. CD34+ cells we obtained in each cord blood sample were very limited. It is not enough to screen the inhibitors concentrations to select the optimal inhibitory concentrations. In the blocking experiments, based on the concentrations used by others and the manufacturer's recommendation, we then determined the inhibitors concentrations by excluding the toxicity of the inhibitors in that concentration, which was determined by clonogenic assay.
Minor comments:
1.The spelling and syntax errors have been checked and corrected.
2.Since the results in figure 1A and B were obtained from two separated and parallel experiments, it is not fitness to combine two figures.
