Isolation, Characterization and Osteogenic Potential of Mouse Digit Tip Blastema Cells in Comparison with Bone Marrow-Derived Mesenchymal Stem Cells In Vitro.

OBJECTIVES: Limb regeneration mediated by blastema cells (BlCs) in mammals is limited to the digit tips of neonates. Due to the lack of access to BlCs in adults and the difficulty in isolating and expanding BlCs from neonates, the use of a cellular population with similar features of BlCs would be a valuable strategy to direct a non-regenerative wound towards regeneration. In this study, we have initially isolated and cultured BlCs, and explored their characteristics in vitro. Next, we compared the capability of bone marrow-derived mesenchymal stem cells (BM-MSCs) as an alternative accessible cell source to BlCs for regeneration of appendages.

MATERIALS AND METHODS: In this experimental study, BM-MSCs were isolated from BM and we obtained BlCs from the neonatal regenerating digit tip of C57B/6 mice. The cells were characterized for expressions of cell surface markers by flow cytometry. Quantitative-reverse transcription polymerase chain reaction (qRT-PCR) and lineage-specific staining were used to assess their ability to differentiate into skeletal cell lineages. The colony forming ability, proliferation, alkaline phosphatase (ALP) activity, calcium content, and osteogenic gene expression were evaluated in both BMMSCs and BlCs cultures at days 7, 14, and 21.

RESULTS: qRT-PCR analysis revealed that the cells from both sources readily differentiated into mesodermal lineages. There was significantly higher colony forming ability in BM-MSCs compared to BlCs (P<0.05). Alizarin red staining (ARS), calcium, and the ALP assay showed the same degree of mineral deposition in both BlCs and BM-MSCs. Gene expression levels of osteblastic markers indicated similar bone differentiation capacity for both BlCs and BM-MSCs at all time-points.

CONCLUSIONS: Characteristics of BlCs in vitro appear to be similar to BM-MSCs. Therefore, they could be considered as a substitute for BlCs for a regenerative approach with potential use in future clinical settings for regenerating human appendages.