Transplantation of spermatogonial stem cells isolated from leukemic mice restores fertility without inducing leukemia

Abstract

More than 70% of patients survive childhood leukemia, but chemotherapy and radiation therapy cause irreversible impairment of spermatogenesis. Although autotransplantation of germ cells holds promise for restoring fertility, contamination by leukemic cells may induce relapse. In this study, we isolated germ cells from leukemic mice by FACS sorting. The cell population in the high forward-scatter and low side-scatter regions of dissociated testicular cells from leukemic mice were analyzed by staining for MHC class I heavy chain (H-2K/H-2D) and for CD45. Cells that did not stain positively for H-2K/H-2D and CD45 were sorted as the germ cell-enriched fraction. The sorted germ cell-enriched fractions were transplanted into the testes of recipient mice exposed to alkylating agents. Transplanted germ cells colonized, and recipient mice survived. Normal progeny were produced by intracytoplasmic injection of sperm obtained from recipient testes. When unsorted germ cells from leukemic mice were transplanted into recipient testes, all recipient mice developed leukemia. The successful birth of offspring from recipient mice without transmission of leukemia to the recipients indicates the potential of autotransplantation of germ cells sorted by FACS to treat infertility secondary to anticancer treatment for childhood leukemia.

Figure 1

Flow cytometric analysis of C1498 cells (A and C) and germ cells (B and D) from normal C57BL/6 mice. Gates for FACS were set for FSC and SSC (A and B). G1 comprises the FSC^high and SSC^low fractions. The staining profiles of CD45 compared with H-2K^b/H-2D^b for the G1 cell fraction of C1498 cells (C) and germ cells (D) from normal C57BL/6 mice are shown with quadrant statistics. None of the C1498 cells in the FSC^high and SSC^low fractions were in the CD45^– and H-2K^b/H-2D^b– fraction, whereas 93.5% of the germ cells were in the CD45^– and H-2K^b/H-2D^b– fraction.

Figure 2

Immunostaining of germ cells before isolation (A) and of the germ cell–enriched fraction (B) with the TRA98 monoclonal antibody specific for nuclei of germ cells from spermatogonia to spermatids. After isolation, the germ cell–enriched fraction only consisted of germ cells. Scale bars: 20 μm.

Figure 3

Isolation of the germ cell–enriched fraction and the leukemic fraction from leukemic mice. (A and B) Flow cytometric analysis of the G1 fraction of germ cells from C1498-inoculated mice. The H-2K^b/H-2D^b– and CD45^– fraction composes the germ cell–enriched fraction (G2), and the H-2K^b/H-2D^b+ and CD45⁺ fraction composes the leukemic cell fraction (G3). Each fraction (G2 and G3) was isolated by FACS. (C) Survival after intraperitoneal injection of cell fractions sorted by FACS. All mice injected with the germ cell–enriched fraction (G2) survived without onset of leukemia for 300 days (n = 12) whereas all mice injected with the leukemic cell fraction (G3) developed terminal signs of leukemia within 40 days (n = 12).

Figure 4

Histologic analysis of intraperitoneally injected mice. Histologic analysis of mice injected with the germ cell–enriched fraction that survived for 300 days showed no signs of leukemia (A, D, and G) whereas histologic analysis of mice injected with the leukemic cell fraction showed infiltration of leukemic cells to the bone marrow (B and E); hemorrhagic ascites also contained leukemic cells (H). (C, F, and I) Control mice. (A–C) H&E-stained bone marrow. (D–F) May-Giemsa–stained bone marrow. (G–I) May-Giemsa–stained peritoneal exudates. Scale bars: 100 μm (A–C); 20 μm (D–I).

Figure 5

Survival after transplantation of sorted germ cells (dashed line, n = 6) or unsorted germ cells (solid line, n = 6) from C1498-inoculated GFP mice into seminiferous tubules. All mice receiving unsorted germ cells developed terminal signs of leukemia within 25 days.

Figure 6

Transplantation of germ cells derived from the testes of leukemic GFP mice into busulfan-treated tubules. (A and B) Cross-sections of a testis at 2 weeks after the transplantation of unsorted germ cells show infiltration by leukemic cells (H&E). (C) Transplanted testis at 8 weeks after transplantation of the germ cell–enriched fraction sorted from the testes of leukemic GFP mice and (D) the corresponding fluorescence image. (E) Seminiferous tubules dissected from a transplanted testis and (F) the corresponding fluorescence image. Donor germ cells (green) colonized in recipient seminiferous tubules. (G) Differentiated germ cells from a transplanted testis. (H and I) Cross-sections of a recipient testis transplanted with the sorted germ cell–enriched fraction. (H) Hematoxylin stain. (I) Fluorescence image. Scale bars: 1 mm (A, C–F); 100 μm (B, H, and I); 20 μm (G).

Figure 7

Sperm from transplanted testes support normal development. (A) Normal progeny produced by intracytoplasmic injection of GFP sperm obtained from transplanted recipient testes. (B) Corresponding fluorescence image. Because hemizygous GFP transgenic mice were used, approximately half of the pups showed green fluorescence.

Figure 8

Kill curve of C1498 leukemia cells in mice. Six mice were injected with each indicated amount of leukemic cells, and the number of days of survival was plotted.