Mesenchymal Stem Cells Maintain Long-Term In Vitro Stemness During Explant Culture

The advantage of mesenchymal stem cells (MSC) in view of cell and/or tissue replacement after transplantation and their prolonged clinical use raises heavy debates not only in the fields of tissue engineering and regenerative medicine to date. Explant culture of umbilical cord (UC) tissue pieces for more than 190 days demonstrated a similar morphology and proliferation rate of outgrowing MSC as compared to UC tissue cultured for 15 days. Flow cytometric analysis revealed the expression of the typical UC-MSC markers CD73, CD90, and CD105 with concomitant absence of CD14, CD31, CD34, and CD45 in all MSC populations. Moreover, subculture of these long-term tissue-derived MSC exhibited nearly identical population doublings and cell cycle distributions and demonstrated the typical MSC surface markers expression until passage 10 in all different explant cultures. Stem cell-like characteristics were also maintained throughout the long term MSC explant cultures, including telomerase activity and the potential to differentiate along the adipogenic, chondrogenic and osteogenic lineage. In contrast, subculture of MSC for more than 10 passages in the absence of the UC tissue microenvironment was uniformly associated with significantly reduced population doublings, cell cycle accumulation in G(0)/G(1), increased senescence and a diminished expression of MCS markers indicating a progressive loss of stemness in all cultures. Together, these findings demonstrated that the stem cell characteristics of MSC can be maintained during long term in vitro culture in the presence of the originating tissue pieces suggesting that the corresponding tissue provides a microenvironment which is essential for keeping MSC in a stem cell-like state.