Persistent heterogeneity of germ cells in stemness and proliferation potential under uniform culture conditions is compatible with a cell-autonomous fate specification model. (A) Environment deterministic and cell-autonomous fate specification models predict homogeneous and heterogeneous composition of SSC progeny, respectively, in a uniform environment. (A, left) In the environment deterministic model, self-renewal and differentiation are controlled by distinct environmental cues (red and blue), which result in homogeneous yet distinct fate outcomes in the red and the blue area. (A, right) In the cell-autonomous fate specification model, occurrence of self-renewal and differentiation does not depend on differential environmental cues, and both SSCs and differentiating progeny can be generated in a uniform environment (gray). (B) Transplantation of germ cells that have been cultured for 20 mo into sterile testes shows that only 16.5 ± 3.9% (mean ± SEM, n = 6) of the population are SSCs, and that 83.5% do not possess stemness. Tracing of sorted single germ cells in culture for 6 mo shows that 79.0 ± 4.1% (mean ± SEM, n = 84 in three indepenent experiments) of the cells perish within a month after transient proliferation, and only 21.0% of the single cells can continue proliferate for at least 6 mo. (C) Dying germ cells show condensed cell bodies and are positive for TUNEL staining. The arrowhead points to a healthy TUNEL-negative germ cell that remains flattened on the culture substrate. Bar, 20 µm.