Changes in the hepatic differentiation potential of human mesenchymal stem cells aged in vitro

BACKGROUND: Due to their multipotency and ability for self-renewal, human umbilical cord-derived mesenchymal stem cells (hUC-MSCs) hold great promise for generating hepatocytes. Previous research has successfully generated hepatocytes from early-passage [i.e., passage (P)3] hUC-MSCs; however, the populations of early-passage cells are limited, and these cells cannot produce sufficient functional hepatocytes for large-scale application in clinical therapy. Thus, a thorough investigation of the hepatic differentiation potential of in vitro-aged hUC-MSCs is needed. METHODS: hUC-MSCs were passaged in vitro and subcultured every 3 days up to P8, and their morphology, proliferative capacity, liver-specific marker expression, and liver function at the end of each passage were analyzed. The efficiency of the hepatogenic differentiation of hUC-MSCs driven by a functional hit 1 (FH1)-based strategy at different passages was also evaluated. RESULTS: The in vitro-aged hUC-MSCs gradually displayed morphological inhomogeneity, had reduced proliferative capability, and exhibited senescent properties while maintaining adipogenic and osteogenic differentiation potential. Additionally, senescence also decreased the expression of messenger RNA (mRNA) levels in albumin (ALB) and alpha 1-antitrpsin (A1AT) in these cells and their relative protein expression, which is the marker of a mature hepatocyte. The liver function of the in vitro-aged hUC-MSCs also deteriorated gradually. Finally, the percentage of hepatocyte-like cells (HLCs) generated from in vitro-aged hUC-MSCs reduced significantly, and the mature hepatocyte functions, such as ALB secretion, glycogen synthesis, low-density lipoprotein (LDL) intake, and indocyanine green (ICG) uptake, also changed. CONCLUSIONS: hUC-MSCs possess mature hepatocytes’ specific markers and functions, which change gradually as they undergo cell senescence. Due to the loss of these properties within in vitro subcultures, the hepatic differentiation efficiency of in vitro-aged hUC-MSCs decreased dramatically in the late passage (P8). The current study provides valuable information can inform future research on liver disease.