The ease of culture expansion of unrestricted somatic stem cells (USSCs) represents one of their primary advantages in clinical strategies. However, genetic alterations during culture expansion undoubtedly affect their therapeutic potential. Telomere shortening with aging is another factor that leads to aberrant stem cell functioning, interfering with potential therapeutic designs. This study evaluates the effect of cell density versus passaging number on the proliferation rate of cord blood (CB)-USSCs, reflected on the telomere length, pluripotent transcription factors expression, and differentiation potential. Methodology: CB-USSCs were cultured at seeding densities of 5000, 500, 50, 5 cells/cm2. Cells from different passages of each seeding density were subjected to pluripotency genes (Oct4, Sox2, Nanog, klf4, c-Myc) and PDGFRa gene expression analysis, measurement of absolute telomere length by real-time PCR, and induction of differentiation into osteogenic, adipogenic, and chondrogenic lineages. Proliferation rate was expressed as population doubling (PD) and cumulative PD (CPD). Results: USSCs from earlier passages (P7) cultured at 5000 cells/cm2 showed the highest telomere length with high expression of pluripotency, and proliferation genes which decreased gradually with passaging till reaching their lowest level at P11 Moreover, their PD at P7 was 3 and CPD (P7-P11) was 12.8. USSCs cultured at 5 cells/cm2 showed PD 12.9 at P7, with a higher expression of gene that plays an important role in proliferation (PDGFRa) than that of 5000 cells/cm2 at P7. Differentiation potentiality of 5000 cells/ cm2 at P7 was high with loss of differentiation at P11, while differentiation potentiality of 5 cells/ cm2 at P7 was much lower than that of 5000 cells/ cm2 at same passage.