This study aimed to investigate whether a representative adult stem cell/precursor cell isolation method (the sphere-forming assay) could isolate cells with differences of telomere length, telomerase activity, and characteristics reflecting senescence. The sphere-forming assay was performed to obtain precursors from cultured sixth passage (P6) human corneal endothelial cells (CECs). P6 and P7 cultured CECs were used as the controls. Telomere length, telomerase activity, and senescence-associated factors were evaluated in precursors and controls. Precursors obtained from the spheres had longer telomeres and higher telomerase activity than cultured P6 cells. Strong positive staining for senescence-associated beta-galactosidase activity was detected in P6 and P7 cultured CECs, whereas little or no staining was detected in the precursors within spheres obtained from P6-cultured CECs or their progeny. The progeny of spheres derived from cultured CECs were small regular cells that grew at a higher density and contained more 5-bromo-2'-deoxyuridine-incorporating cells compared with the parental cultured cells. These findings indicate that the sphere-forming assay enriches precursors with longer telomeres, higher telomerase activity, and younger progeny than the original cells. Thus, the sphere-forming assay may contribute to obtaining the young cells needed for regenerative medicine.