Fig. 5. Neural crest-derived and mesoderm-derived periosteal cells have distinct
proliferation and osteogenic differentiation potentials. (A)
Skeletal progenitor cells from the mandibular periosteum and GFP-positive
tibial periosteum were co-cultured for 0, 3, 5 and 7 days; cell nuclei were
labeled with Hoechst and viewed using fluorescent imaging. (B)
Quantification of the co-culture showed a linear increase in the number of
GFP-positive mesoderm-derived cells and only a minor increase in the number of
neural crest-derived cells. (C) BrdU incorporation assay at 3, 5 and 7
days showed higher proliferation rates for mesoderm-derived cells at all time
points. (D,E) Alizarin Red staining of mesoderm-derived and
neural crest-derived skeletal progenitor cell cultures after 10, 12 and 14
days in osteogenic differentiation media. (F) Quantification of
Alizarin Red mineralization showed a statistically significant increase in the
amount of mineralized matrix in neural crest-derived samples. (G)
qRT-PCR performed on RNA isolated from cell populations after 4 days in vitro.
Neural crest-derived cells showed an increase in the expression of
Runx2 and Col1a. (H) qRT-PCR performed on RNA
isolated from cell populations after 10 days in vitro demonstrated that neural
crest-derived cells showed an increase in the expression of all osteogenic
markers, including Runx2, osteopontin (op), Col1a and
osteocalcin (oc). *P
0.01.