Hum. Reprod. Advance Access originally published online on August 27, 2007
Human Reproduction 2007 22(10):2797-2798; doi:10.1093/humrep/dem213
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Letters to the Editor |
Reply: Isolation of germ cells from leukaemic cells
1 Research Centre for Reproduction and Genetics, University Hospital and Medical School, Vrije Universiteit Brussel, Laarbeeklaan 101, B-1090 Brussels, Belgium 2 Centre for Reproductive Medicine, University Hospital and Medical School, Vrije Universiteit Brussel, Laarbeeklaan 101, B-1090 Brussels, Belgium
3 Correspondence address. Tel: +32 2 477 6673; Fax: +32 2 477 6692; E-mail: mieke.geens{at}uzbrussel.be
We would like to thank Dr Fujita and colleagues for their letter to the editor regarding our recent article (Geens et al., 2007
). We have read their comments with interest and we certainly agree that the separation of germ cells from cancer cells is a matter under debate.
In their letter, the authors state that the fact that none of the recipients (n = 12) in their study developed leukaemia proves that fluorescence-activated cell sorting (FACS) is sufficient to reliably separate tumour cells from testicular cells. We cannot agree with this assumption because, as can be seen in Table 2 of our article, not all mice developed tumours when only a low number of malignant cells was injected. The lower the number of cells injected, the lower chances of developing tumours, but even after injecting between 5 and 39 malignant cells, still more than 40% of mice developed malignancy. Moreover, after checking our data from the transplantations with the sorted fractions, we observed that out of 20 mice, the first 15 mice did not develop any malignancy but in the 16th mouse, a tumour was observed (data not presented as such in our paper). We therefore conclude that sorted cell fractions should be analysed extremely carefully to avoid even minimal contamination, since the exact number of cancer cells able to induce malignancy is unknown.
As we already mentioned in the discussion of our manuscript, non-specific aggregation of cancer cells to germ cells is possible (Jahnukainen et al., 2006). We therefore agree that it would not be a good option to try to separate germ cells from leukaemic cells, solely relying on positive selection for germ cell markers. In the murine set-up, we used magnetic-activated cell sorting for positive selection of CD49f or 
6-integrine expressing cells (germ cells). However, the aim of this step was not really to remove the malignant cells, but to enrich spermatogonial stem cells in the samples, thereby allowing a faster and more efficient FACS where only cells that were negative for H-2Kb and at the same time positive for CD49f were selected. If a malignant cell would adhere to a germ cell, this aggregation would be positive for both H-2Kb and CD49f and would not be selected. Even though positive selection for spermatogonial stem cells might not be necessary for the depletion of cancer cells, enrichment of these stem cells might be unavoidable in a clinical set-up, since the success of the spermatogonial stem cell transplantation technique is highly dependent on the number of stem cells injected (Dobrinski et al., 1999).
It is true that CD49f is frequently expressed by leukaemic cells, but as mentioned in the article, for both cell lines used in our study, CD49f was not expressed (according to the gates used). In a clinical set-up, we would not advise to use the same common marker for sorting suspensions of individual patients. Immunophenotyping of the malignant clone for each individual patient would be necessary to find the appropriate markers for reliable sorting.
We agree that the gates used in Figs 2A and B and 4A and B might comprise some malignant cells. However, these are not identical to the gates that we used in the experiments. Owing to technical restrictions, we were unable to transfer the co-ordinates of the gates from the computer where the analyses were done to the computer where the pictures were generated. The gates that were set during the sorting and analyses were generated using positive (labelled malignant cells) and negative (labelled testicular cells and unlabelled cells) controls. The gates in the pictures were only added to clarify the sorting parameters. Certainly, the gates in the pictures could even then have been chosen more precisely in order to avoid any confusion for the readership.
In conclusion, we certainly do not want to discourage further research on decontamination of testicular suspensions using FACS. We want to stress, however, that analysis of the sorted fractions should be performed very carefully and with an extremely sensitive test and in an appropriate number of samples. We believe that criteria, as applied for the detection of minimal residual disease should also be used in the isolation of germ cells from contaminated testicular cell suspensions, before even considering a clinical set-up. As discussed in our paper, other techniques like the in vitro culture of spermatogonial stem cells could be applied to reach a more efficient and safer transplantation.
References
Geens M, Van de Velde H, De Block G, Goossens E, Van Steirteghem A, Tournaye H. The efficiency of magnetic-activated cell sorting fluorescence-activated cell sorting in the decontamination of testicular cell suspensions in cancer patients. Hum Reprod (2007) 22:733–742.
Dobrinski I, Ogawa T, Avarbock MR, Brinster RL. Computer assisted image analysis to assess colonization of recipient seminiferous tubules by spermatogonial stem cells from transgenic donor mice. Mol Reprod Dev (1999) 53:142–148.[CrossRef][Web of Science][Medline]
Jahnukainen K, Ehmcke J, Soder O, Schlatt S. Clinical potential and putative risks of fertility preservation in children utilizing gonadal tissue or germline stem cells. Pediatr Res (2006) 59:40R–47R.[CrossRef][Web of Science][Medline]
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