Effect of isolation methodology on stem cell properties and multilineage differentiation potential of human dental pulp stem cells

Abstract

Dental pulp stem cells (DPSCs) are an attractive alternative mesenchymal stem cell (MSC) source because of their isolation simplicity compared with the more invasive methods associated with harvesting other MSC sources. However, the isolation method to be favored for obtaining DPSC cultures remains under discussion. This study compares the stem cell properties and multilineage differentiation potential of DPSCs obtained by the two most widely adapted isolation procedures. DPSCs were isolated either by enzymatic digestion of the pulp tissue (DPSC-EZ) or by the explant method (DPSC-OG), while keeping the culture media constant throughout all experiments and in both isolation methods. Assessment of the stem cell properties of DPSC-EZ and DPSC-OG showed no significant differences between the two groups with regard to proliferation rate and colony formation. Phenotype analysis indicated that DPSC-EZ and DPSC-OG were positive for CD29, CD44, CD90, CD105, CD117 and CD146 expression without any significant differences. The multilineage differentiation potential of both stem cell types was confirmed by using standard immuno(histo/cyto)chemical staining together with an in-depth ultrastructural analysis by means of transmission electron microscopy. Our results indicate that both DPSC-EZ and DPSC-OG could be successfully differentiated into adipogenic, chrondrogenic and osteogenic cell types, although the adipogenic differentiation of both stem cell populations was incomplete. The data suggest that both the enzymatic digestion and outgrowth method can be applied to obtain a suitable autologous DPSC resource for tissue replacement therapies of both bone and cartilage.

How has dental pulp stem cells isolation been conducted? A scoping review

“…29 This technique is based on the outgrowth of cells from small tissue fragments, and it allows the isolation of a relatively pure population of pulp stem cells. 19 However, a heterogeneous population of cells, achieved with the enzymatic technique, is needed in some cell-based therapy approaches, since it can endure various environmental signals within the tissue 29 .…”

“…18 Few studies have compared the enzymatic and explant techniques for DPSCs isolation. 23,29,31 In a well-designed study, Hilkens and collaborators showed that cells isolated by the enzymatic or the explant techniques had the same proliferating rate and colony formation capacity, and could successfully differentiate into adipogenic, chrondrogenic and osteogenic cell types, indicating that both isolation methodologies could be applied to obtain suitable autologous DPSCs. 29 One particular aspect of the explant technique is the size of tissue fragments.…”

“…23,29,31 In a well-designed study, Hilkens and collaborators showed that cells isolated by the enzymatic or the explant techniques had the same proliferating rate and colony formation capacity, and could successfully differentiate into adipogenic, chrondrogenic and osteogenic cell types, indicating that both isolation methodologies could be applied to obtain suitable autologous DPSCs. 29 One particular aspect of the explant technique is the size of tissue fragments. According to our results fragment dimensions are not standardized, since 45.5% of the articles did not provide this information and 18.2% only mentioned that tissue fragments were small.…”

What is the clinical applicability of regenerative therapies in dentistry?

“…Two methods are used to perform the isolation of mesenchymal stem cells 13 : 1) Enzymatic digestion; 2) Primary culture or Explant…”

“…enzymes could be aggressive towards these cells. However, both techniques are considered adequate for obtaining stem cells 13 . Once an adequate amount of cells has been obtained, they can be used in SC-BT or safely frozen either in liquid nitrogen for up to 2 years or in a freezer at -80ºC for up to one year 4 .…”