Hum. Reprod. Advance Access originally published online on August 28, 2007
Human Reproduction 2007 22(10):2796-2797; doi:10.1093/humrep/dem212
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Letters to the Editor |
Isolation of germ cells from leukemic cells
Department of Urology, Osaka University Graduate School of Medicine, 2-2 Yamada-oka, Suita 565-0871, Japan
1 Correspondence address. Tel: +81-6-6879-3531; Fax: +81-6-6879-3539; E-mail: akitsuji{at}uro.med.osaka-u.ac.jp
We have read with great interest the article by Geens et al. (2007)
, titled ‘The efficiency of magnetic-activated cell sorting and fluorescence-activated cell sorting in the decontamination of testicular cell suspensions in cancer patients' published on Hum Reprod.
The authors concluded that fluorescence-activated cell sorting (FACS) was neither in a murine nor in a human model sufficient to completely deplete testicular tissue of malignant cells. Specifically, in the human model only one of 11 tumour-tissue suspensions were completely depleted of malignant cells. Their results contradict previous reports by our group (Fujita et al., 2005). We were able to restore fertility in sterile mice by transplantation of spermatogonial stem cells. We demonstrated that none of the recipients developed leukemia after transplantation of tumour-cell-depleted spermatogonial cells, isolated from leukemic mice.
As Geens et al. pointed out, one limitation of our study is the low number of mice (n = 12) included and we agree that further investigations to confirm our preliminary findings are warranted. Furthermore, we did not re-analyse the sorted fraction for presence of tumour cells. However, the fact that none of the recipients developed leukemia in our study as the most convincing clinical outcome variable, supports our hypothesis that FACS is sufficient to reliably separate tumour cells from spermatogonial stem cells.
However, we detected some limitations of the current study: positive selections with surface markers expressed on germ cells harbour the risks of contamination by leukemic cells since leukemic cells may adhere non-specifically to germ cells and may bypass the selection for CD49f. These adherent tumour cells would consequently be isolated as non-tumour cells in gate 1 (R1) using FACS analysis. Therefore, negative selection utilizing surface markers expressed only on leukemic cells is preferred since this prevents the above mentioned mechanism of tumour cell contamination due to adherence of tumour cells to germ cells. Furthermore, CD49f (Stucki et al., 2001) is a frequently expressed surface antigen on human leukemia cells and the isolation of spermatogonial stem cells solely relying on positively for CD49f harbours a high risk of tumour cell contamination. If the enrichment of spermatogonial stem cells ultilizing CD49f as selection criteria is desired, it would be essential to deplete for leukemia cells via negative selection first and in a second step enrich for spermatogonial stem cells via positive selection.
Furthermore, it seems that the limits of gate 2 (R2) were chosen too generously: the cutoff for the H-2Kb axis was 80 on and 
12 on CD49f axis. The spermatogonial stem cell fraction was sorted by utilizing R2 although R2 would contain some EL-4 cells when superimposed on Fig. 2A. Gate ‘R1’ was also set at an approximate intensity of 70 at the HLA-A–C axis in Fig. 4B. However, the non-R1 gate of Fig. 4B would still contain a fair amount of SB cells when superimposed on Fig. 4A. Therefore, by using the gates depicted in Figs. 2A and B and 4A and B, the contamination of spermatogonial stem cells by leukemic cells is an inevitable consequence. As we suggested in our previous paper, it is of utmost importance to set the gate in a very restricted way to prevent contamination. It is well known that some subtypes of human leukemia cells express MHC-class I in low abundance which would result in a high risk of tumour cell contamination if isolation is based on only one (Fujita et al., 2006).
Our group isolated spermatogonial stem cells by setting gates for ‘MHC-class I-negative and CD45-negative region’ among the cell population of forward scatter (FCS)high and side scatter (SSC)low. We detected some leukemic cells in the MHC-class I-negative and CD45-negative region, if the additional gates, ‘FSChigh and SSClow’, were not used. The enhancement of surface marker expression by interferon gamma would be necessary. Alternatively, additional surface antigens should be used to reliably separate spermatogonial stem and leukemia cells.
Overall the use of spermatogonial stem cell separation from malignant cells by FACS should not be discouraged. Rather additional studies in this regard are warranted. One future direction to circumvent this problem could be the development of in vitro proliferation of human spermatogonial stem cells: after the isolation of spermatogonial stem cells by FACS, an isolated single stem cell could form a colony, from which only one cell would be chosen for in vitro proliferation with no risk of tumour cell contamination.
References
Fujita K, Ohta H, Tsujimura A, Takao T, Miyagawa Y, Takada S, Matsumiya K, Wakayama T, Okuyama A. Transplantation of spermatogonial stem cells isolated from leukemic mice restores fertility without inducing leukaemia. J Clin Invest (2005) 115::1855–1861.[CrossRef][Web of Science][Medline]
Fujita K, Tsujimura A, Miyagawa Y, Kiuchi H, Matsuoka Y, Takao T, Takada S, Nonomura N, Okuyama A. Isolation of germ cells from leukemia and lymphoma cells in a human in vitro model: potential clinical application for restoring human fertility after anticancer therapy. Cancer Res (2006) 66:11166–11171.
Geens M, Van de Velde H, De Block G, Goossens E, Van Steirteghem A, Tournaye H. The efficiency of magnetic-activated cell sorting and fluorescence-activated cell sorting in the decontamination of testicular cell suspensions in cancer patients. Hum Reprod (2007) 22:733–742.
Stucki A, Rivier AS, Gikic M, Monai N, Schapira M, Spertini O. Endothelial cell activation by myeloblasts: molecular mechanisms of leukostasis and leukemic cell dissemination. Blood (2001) 97:2121–2129.
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