Functional assessment of spermatogonial stem cell purity in experimental cell populations

Abstract

Historically, research in spermatogonial biology has been hindered by a lack of validated approaches to identify and isolate pure populations of the various spermatogonial subsets for in-depth analysis. In particular, although a number of markers of the undifferentiated spermatogonial population have now been characterized, standardized methodology for assessing their specificity to the spermatogonial stem cell (SSC) and transit amplifying progenitor pools has been lacking. To date, SSC content within an undefined population of spermatogonia has been inferred using either lineage tracing or spermatogonial transplantation analyses which generate qualitative and quantitative data, respectively. Therefore, these techniques are not directly comparable, and are subject to variable interpretations as to a readout that is representative of a 'pure' SSC population. We propose standardization across the field for determining the SSC purity of a population via use of a limiting dilution transplantation assay that would eliminate subjectivity and help to minimize the generation of inconsistent data on 'SSC' populations. In the limiting dilution transplantation assay, a population of LacZ-expressing spermatogonia are selected based on a putative SSC marker, and a small, defined number of cells (i.e. 10 cells) are microinjected into the testis of a germ cell-deficient recipient mouse. Using colony counts and an estimated colonization efficiency of 5%; a quantitative value can be calculated that represents SSC purity in the starting population. The utilization of this technique would not only be useful to link functional relevance to novel markers that will be identified in the future, but also for providing validation of purity for marker-selected populations of spermatogonia that are commonly considered to be SSCs by many researchers in the field of spermatogenesis and stem cell biology.

Keywords: Limiting dilution; Spermatogonial stem cell; Transplantation.

Fig. 1

A limiting dilution transplantation assay to determine SSC content in undefined testis cell populations. Testis cells are collected from a donor mouse harboring a ubiquitously expressed LacZ transgene. A sub-population of cells are then isolated based on expression of a marker of interest using cell sorting strategies. Selected cells are subjected to a limiting dilution series, creating populations of 1000 (LD1000), 100 (LD100) and 10 (LD10) cells. These populations are transplanted into the testes of busulfan treated mice. At >2 months post-transplantation, LacZ expressing donor-derived colonies are visualized in recipient testes following X-gal staining. By factoring in a colonization efficiency of 5%, it is expected that donor populations containing pure SSCs would produce colonies not only in LD1000 and LD100 transplanted testes, but also in a subset of LD10 treated testes. Contrastingly, colony number would be expected to be reduced in LD100 and absent in LD10 transplanted testes if the donor population was a heterogeneous mix of SSC and transit amplifying progenitor spermatogonia. A quantitative value reflecting SSC purity in the donor population can be calculated using the formula: [# cells transplanted / (# colonies of donor-derived spermatogenesis/5% colonization efficiency)] = x, where 1 in every x cells is an SSC.