Stem Cells from Cryopreserved Human Dental Pulp Tissues Sequentially Differentiate into Definitive Endoderm and Hepatocyte-Like Cells <i>in vitro</i>

TLDR

A novel tissue cryopreservation protocol has been developed to safely preserve various autologous stem cell sources. This study characterizes stem cells from long‑term cryopreserved dental pulp tissues and evaluates their in‑vitro differentiation into definitive endoderm and hepatocyte‑like cells. Human dental pulp tissues from extracted wisdom teeth were cryopreserved for at least a year, thawed, compared to fresh counterparts, then differentiated into definitive endoderm with Activin A/Wnt3a for six days and further into hepatocyte‑like cells with growth factors until day 30. The cryopreserved cells displayed similar growth and mesenchymal stem cell traits as fresh cells, and during differentiation they adopted hepatocyte‑like morphology, upregulated DE and hepatocyte markers, and exhibited detoxification and glycogen storage, confirming that hDPSCs‑cryo can generate functional hepatocyte‑like cells in vitro and offering a valuable autologous source for tissue engineering.

Abstract

We previously described a novel tissue cryopreservation protocol to enable the safe preservation of various autologous stem cell sources. The present study characterized the stem cells derived from long-term cryopreserved dental pulp tissues (hDPSCs-cryo) and analyzed their differentiation into definitive endoderm (DE) and hepatocyte-like cells (HLCs) in vitro. Human dental pulp tissues from extracted wisdom teeth were cryopreserved as per a slow freezing tissue cryopreservation protocol for at least a year. Characteristics of hDPSCs-cryo were compared to those of stem cells from fresh dental pulps (hDPSCs-fresh). hDPSCs-cryo were differentiated into DE cells in vitro with Activin A as per the Wnt3a protocol for 6 days. These cells were further differentiated into HLCs in the presence of growth factors until day 30. hDPSCs-fresh and hDPSCs-cryo displayed similar cell growth morphology, cell proliferation rates, and mesenchymal stem cell character. During differentiation into DE and HLCs in vitro, the cells flattened and became polygonal in shape, and finally adopted a hepatocyte-like shape. The differentiated DE cells at day 6 and HLCs at day 30 displayed significantly increased DE- and hepatocyte-specific markers at the mRNA and protein level, respectively. In addition, the differentiated HLCs showed detoxification and glycogen storage capacities, indicating they could share multiple functions with real hepatocytes. These data conclusively show that hPDSCs-cryo derived from long-term cryopreserved dental pulp tissues can be successfully differentiated into DE and functional hepatocytes in vitro. Thus, preservation of dental tissues could provide a valuable source of autologous stem cells for tissue engineering.