Germline stem cells: toward the regeneration of spermatogenesis

Abstract

Improved therapies for cancer and other conditions have resulted in a growing population of long-term survivors. Infertility is an unfortunate side effect of some cancer therapies that impacts the quality of life of survivors who are in their reproductive or prereproductive years. Some of these patients have the opportunity to preserve their fertility using standard technologies that include sperm, egg, or embryo banking, followed by IVF and/or ET. However, these options are not available to all patients, especially the prepubertal patients who are not yet producing mature gametes. For these patients, there are several stem cell technologies in the research pipeline that may give rise to new fertility options and allow infertile patients to have their own biological children. We will review the role of stem cells in normal spermatogenesis as well as experimental stem cell-based techniques that may have potential to generate or regenerate spermatogenesis and sperm. We will present these technologies in the context of the fertility preservation paradigm, but we anticipate that they will have broad implications for the assisted reproduction field.

Keywords: Male fertility; male infertility; regenerative medicine; spermatogonial stem cells; stem cells.

Figure 1

Standard and experimental options for preserving male fertility. Top, sperm obtained by ejaculation or surgical retrieval from the testes or epididymides are competent to fertilize oocytes using assisted reproductive techniques including intrauterine insemination (IUI), in vitro fertilization (IVF) or IVF with intracytoplasmic sperm injection (ICSI)) that are standard in most fertility clinics. These options are not available to prepubertal boys who are not producing sperm or to adult azoospermic men. Bottom, testis tissue obtained via biopsy from prepubertal boys contains SSCs that can produce sperm in the context of the intact tissue by xenotransplant, organ culture or autologous transplantation back into the individual (orange boxes). Sperm retrieved from cultured or transplanted tissue can be used for ICSI. Cells in suspension obtained from biopsied testicular tissue can be transplanted back into the endogenous seminiferous tubules of the patient (blue boxes). SSCs in the suspension can regenerate spermatogenesis and, in some cases, fertility. For infertile individuals who did not preserve germs cells before gonadotoxic therapy, induced pluripotent stem cells (IPSCs) may be produced from his somatic cells (e.g., skin or blood) to differentiate into transplantable germ cells (PGCs or SSCs) or haploid germ cells that can be used for ICSI (red boxes). Excerpted with permission from Clark AT, Phillips BT and Orwig KE 2011 NATURE MEDICINE 17:1564–1565.

Figure 2

Human spermatogonial stem cells and spermatogenesis. (A) Testes are comprised of seminiferous tubules that start and end at the rete testis. (B) Cut-out of the basement membrane of the seminiferous epithelium. (B and C) The basement membrane of the seminiferous epithelium contains undifferentiated (A_dark and A_pale) spermatogonia and differentiating Type B spermatogonia. Type B spermatogonia give rise to primary spermatocytes that enter meiosis and migrate off the basement membrane. Subsequent meiotic divisions and morphogenesis give rise to secondary spermatocytes, spermatids and the terminally differentiated spermatozoa that are released into the lumen of the seminiferous tubules

Figure 3

Ultrasound-guided rete testis injections. For SSC transplantation into larger animals, including nonhuman primates, (A) ultrasound is used to visualize the rete testis (echo-dense structure). (B and C) The injection needle is inserted under ultrasound guidance through the scrotal skin into the rete testis space, which is continuous with the seminiferous tubules. (C) Positive pressure is applied to the needle so the cells are slowly injected into the rete testis space and seminiferous tubules.

Figure 4

Experimental techniques to assay human spermatogonia. (A,B and C) Expression of spermatogonia markers PLZF (A), SALL4 (B) and PGP9.5 (C) is limited to germ cells located on the basement membrane of human seminiferous tubules. Thus, they are reliable markers to screen test cell populations for human spermatogonia. DAPI (blue) stains all cell nuclei. Scale bar = 50µm. (D) Human to nude mouse xenotransplantation assay. Human testicular tissue is made into a cell suspension and then transplanted into the testis of busulfan-treated infertile nude mice. Two months following the transplantation, the testes are recovered, the tunica is removed and the seminiferous tubules are gently dispersed to make a whole mount. The tubules are then stained with anti-primate antibody (122) to recognize the colonies of human spermatogonia (green).