Testicular Niche Required for Human Spermatogonial Stem Cell Expansion

Abstract

Prepubertal boys treated with high-dose chemotherapy do not have an established means of fertility preservation because no established in vitro technique exists to expand and mature purified spermatogonial stem cells (SSCs) to functional sperm in humans. In this study, we define and characterize the unique testicular cellular niche required for SSC expansion using testicular tissues from men with normal spermatogenesis. Highly purified SSCs and testicular somatic cells were isolated by fluorescence-activated cell sorting using SSEA-4 and THY1 as markers of SSCs and somatic cells. Cells were cultured on various established niches to assess their role in SSC expansion in a defined somatic cellular niche. Of all the niches examined, cells in the SSEA-4 population exclusively bound to adult testicular stromal cells, established colonies, and expanded. Further characterization of these testicular stromal cells revealed distinct mesenchymal markers and the ability to undergo differentiation along the mesenchymal lineage, supporting a testicular multipotent stromal cell origin. In vitro human SSC expansion requires a unique niche provided exclusively by testicular multipotent stromal cells with mesenchymal properties. These findings provide an important foundation for developing methods of inducing SSC growth and maturation in prepubertal testicular tissue, essential to enabling fertility preservation for these boys.

Spermatogonial stem cells and spermatogenesis in mice, monkeys and men

“…ITGA6 expression is not restricted to SSCs or even germ cells, but the entire population of functional SSCs can be recovered and are significantly enriched in the ITGA6+ fraction of rodent (Shinohara et al, 2000) and human testis cells (Valli et al, 2014). SSEA4 has not been used to isolate mouse SSCs, but is expressed by undifferentiated spermatogonia in monkey and human testes and has been used effectively to isolate transplantable SSCs (Zheng et al, 2014; Muller et al, 2008; Izadyar et al, 2011; Eildermann et al, 2012b; Smith et al, 2014). CD9 is expressed by a subpopulation of MAGEA4+ spermatogonia in human testes and can be used to isolate transplantable stem cells (Zohni et al, 2012).…”

Cryopreservation of testicular tissue before long-term testicular cell culture does not alter in vitro cell dynamics

“…Given the rising concern in recent literature about possible misinterpretation of human SSC propagation data and the lack of a true SSC marker (13)(14)(15)(16), we opted for the combined approach for the unequivocal detection of SSCs as proposed by Eildermann et al (13). Specifically, we used VASA as a reliable marker for the detection of human germ cells in vitro (15), together with the in vitro spermatogonial marker ubiquiting carboxyl-terminal esterase L1 (UCHL1) (5)(6)(7)17).…”

Experimental methods to preserve male fertility and treat male factor infertility

“…Cultured rodent SSCs can be maintained in long-term culture with exponential expansion in numbers and retain their biological potential to produce spermatogenesis and restore fertility when transplanted into the testes of infertile recipient mice 70–73 . Langenstroth and colleagues have reported maintaining nonhuman primate SSCs in short-term culture 74 and several groups have reported culturing human SSCs for short- or long-term 75–81,24,82,83 . Each group reporting human SSC culture used different methods to isolate cells for culture, different feeder or matrix substrates, different growth factor cocktails and different methods to assess progress.…”