Umbilical cord matrix stem cells (UCMSC) have generated great interest in various therapeutic approaches, including liver regeneration. This article aims to analyze the specific characteristics and the potential occurrence of premalignant alterations of UCMSC during long-term expansion, which are important issues for clinical applications. UCMSC were isolated from the umbilical cord of 14 full-term newborns and expanded in vitro until senescence. We examined the long-term growth potential, senescence characteristics, immunophenotype and multilineage differentiation capacity of these cells. In addition, their genetic stability was assessed through karyotyping, telomerase maintenance mechanisms and analysis of expression and functionality of cell cycle regulation genes. The tumorigenic potential was also studied in immunocompromised mice. In vitro, UCMSC reached up to 33.7 ± 2.1 cumulative population doublings before entering replicative senescence. Their immunophenotype and differentiation potential, notably into hepatocyte-like cells, remained stable over time. Cytogenetic analyses did not reveal any chromosomal abnormality and the expression of oncogenes was not induced. Telomere maintenance mechanisms were not activated. Just as UCMSC lacked transformed features in vitro, they could not give rise to tumors in vivo. UCMSC could be expanded in long-term cultures while maintaining stable genetic features and endodermal differentiation potential. UCMSC therefore represent safe candidates for liver regenerative medicine.