Figure 2

Osteoblastogenesis investigations. (a–g) Bone marrow stromal cell (BMSC) in vitro osteoblastogenesis assays. 11 week-old male SPF & GF mice were euthanized; bone marrow harvested; BMSCs isolated for in vitro assays. (a) Cell expansion assay (n = 4/gp): cell numbers over time in culture. (b–e) BMSC differentiation potential assay (untreated day-4 pre-confluent cultures were harvested for qRT-PCR analysis) to assess intrinsic differentiation potential (n = 4/gp). (b) Pparg mRNA assessed as a marker of adipogenic potential. (c) Col2a1 mRNA assessed as a marker of chondrogenic potential. (d) Runx2 and (e) Sp7(Osterix) mRNA assessed as markers of osteoblastogenic potential. Relative quantification of mRNA was performed via the comparative C _T method (ΔΔCT); Gapdh was utilized as an internal control gene; data expressed as fold difference relative to SPF. (f,g) von Kossa mineralization assay (21 day mineralization treatment) (n = 4/gp). (f) Representative von Kossa stained culture images. (g) Mineralization area per well area. (a–g) BMSC assays carried out in duplicate (two technical replicate) cultures; n-values represent biological replicates per group. (h–j) Commensal microbiota in vivo regulation of osteoblastogenesis. 11 to 12 week-old male SPF & GF mice were euthanized; tissues were harvested. (h,i) RNA was isolated from marrow (n = 4/gp), calvaria (n = 4/gp), liver (n = 6/gp) for qRT-PCR analysis of candidate osteogenic genes. (h) Bglap(Osteocalcin) mRNA assessed as a marker of mature osteoblast function, and (i) Igf1 mRNA assessed as a critical osteoblastic signaling factor. Relative quantification of mRNA was performed via the comparative C _T method (ΔΔCT); Gapdh was utilized as an internal control gene; data expressed as fold difference relative to SPF. (j) Serum was isolated from whole blood (n = 10/gp); ELISA analysis of IGF1 levels. Data reported as mean ± SEM. *p < 0.05 vs. SPF; **p < 0.01 vs. SPF.