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

Abstract

More than 70% of patients survive childhood cancer, but chemotherapy and radiation therapy may cause irreversible impairment of spermatogenesis. To treat infertility secondary to anticancer treatment for childhood cancer, we have developed a procedure to isolate germ cells from leukemic mice by fluorescence-activated cell sorting with two surface markers, and transplantation of isolated germ cells successfully restored fertility without inducing leukemia. In the present study, we analyzed human germ cells and human malignant cells, including five leukemia cell lines and three lymphoma cell lines, by fluorescence-activated cell sorting with antibodies against MHC class I and CD45. Testicular specimens were obtained from a patient who underwent surgery for testicular rupture. In the high forward scatter and low side scatter region, no malignant cells were found in the MHC class I-negative and CD45-negative fraction (the germ cell fraction), with the exception of K562 cells. A total of 39.2% of the germ cells were found in the germ cell fraction. A total of 1.45% of K562 cells were found in the germ cell fraction. Treatment with IFN; induced the expression of MHC class I on K562 cells but not on germ cells and made it possible to isolate germ cells from K562 cells. In conclusion, we isolated human germ cells from malignant cells with two surface markers after treatment with IFN;. Immunophenotyping for each patient will be necessary before isolation and induction of surface marker will be clinically applicable.

Eliminating malignant contamination from therapeutic human spermatogonial stem cells

“…Additionally, viable offspring were generated from the infertile recipients following transplantation of the sorted germ cells (24). Fujita and colleagues followed up this initial report by demonstrating that 7 out of 8 human leukemic cell lines also expressed the cell surface antigens CD45 and MHC class I, and thus these leukemic markers could theoretically be used to separate leukemic cells from testicular cells in humans as well, but this was not assessed experimentally in that study (25). Hermann and coworkers demonstrated the feasibility of removing contaminating leukemic cells from nonhuman primate testis cell suspensions by FACS sorting with THY-1 (spermatogonial marker) and CD45 (leukemia marker) (35).…”

“…To identify potential spermatogonial markers that could be used to isolate/ enrich spermatogonia and were distinct from MOLT-4 cells, our goal was to identify antigens that were expressed by less than 1% of MOLT-4 cells and by 5% or more of human testis cells. CD49f (α6 integrin), CD29 (β1 integrin), CD90 (THY-1), and CD326 (EpCAM) were of particular interest, because these markers have been demonstrated to be expressed on spermatogonia in humans (CD49f, CD90) or in other animal models (25,36,(47)(48)(49)(50). CD49f, CD29, and CD90 were rejected for further consideration, because they had >1% expression on MOLT-4 cells.…”

“…In 2006, Fujita and colleagues demonstrated via flow cytometry that several human leukemic cell lines uniformly expressed cell surface antigens MHC class I and CD45 (25). They then performed FACS on human testicular cells and demonstrated that the MHC class I -/CD45 -fraction contained germ cells (assessed qualitatively by RT-PCR for germ cell markers), suggesting that these cell surface antigens could be used to sort leukemic cells away from germ cells.…”

“…The potential of using SSCs to preserve and restore fertility in patients receiving gonadotoxic therapies has been extensively discussed (23)(24)(25)(26)(27)(28)(29)(30)(31)(32). In theory, testicular cells obtained via biopsy prior to cancer treatment could be cryopreserved and then retransplanted following clinical remission.…”

Spermatogonial stem cell biomarkers: improved outcomes of spermatogonial transplantation in male fertility restoration?

“…This resulted in regeneration of spermatogenesis and fertility restoration in recipient mice without causing transmission of leukemia. This approach has recently been expanded to a human in vitro model [43]. Thus, application of negative biomarkers for SSCs should allow depletion of tumor cells from a testis biopsy, which will be a critical step to assure protection against tumor relapse in the SSC-based fertility restoration scheme.…”