Figure 1.
Characterization of human MSCs by their surface marker expression and their ability to differentiate into mesenchymal lineages.
(a) Bone marrow derived human MSCs express CD13, CD44, CD73, CD90, and CD105 but do not express CD45, CD34, CD14, and CD19; they differentiate into different mesenchymal lineages such as (b) adipogenic, (c) osteogenic and (d) chondrogenic lineages (scale indicated on the figures; n = 8).
Table 1.
Percentage of CD-positive cell populations.
Figure 2.
Hypoxia does not alter MSC phenotype and surface marker expression.
No difference between MCSs maintained under normoxia and those incubated under hypoxia were observed in terms of (a) cell morphology (after 6 h, 24 h, and 2w of incubation; scale indicated on the figures; n = 3) and (b) surface marker expression of the positive-markers CD13, CD44, CD73, CD90, CD105 and the negative-markers CD45, CD34, CD14, and CD19 (normoxia = red; hypoxia = green; n = 6).
Table 2.
Percentage of CD-positive cell populations after 2 week incubation under normoxia.
Table 3.
Percentage of CD-positive cell populations after 2 week incubation under hypoxia.
Figure 3.
Hypoxia induces HIF-1α and HIF-1-target-gene expression.
(a) HIF1A gene expression of human MSCs obtained by real-time PCR (n = 6; unpaired t-test). (b) HIF-1alpha and beta-actin protein expression obtained by immunoblot. (c) Hypoxia-induced HIF-1-target-gene expression of VEGFA, LDHA, GLUT1, and PGK1 (n = 6; 2-weeks data; one sample t-test; dotted-line indicates normalization to gene expression of normoxic cells; *** p<0.001; ** p<0.01; * p<0.05).
Figure 4.
Hypoxia suppresses adipogenic and promotes osteogenic differentiation of human MSCs.
(a) Oil-Red-O stain for the analysis of adipogenesis and von-Kossa stain for the analysis of osteogenesis of MSCs incubated under normoxia (≈18% pO2) or hypoxia (1% pO2) for 4 weeks using either osteogenic or adipogenic differentiation medium (scale indicated on the figures; n = 6). (b) HIF1A, (c) VEGFA, (d) PPARG and (e) RUNX2 gene expression of MSCs incubated under normoxia (≈18% pO2) or hypoxia (1% pO2) for 2 weeks using either osteogenic or adipogenic differentiation medium as obtained by real-time PCR (n = 6; unpaired t-test; dotted-line indicates normalisation to gene expression of undifferentiated cells; * p<0.05). (f) Analysis of osteogenesis by von-Kossa stain of MSCs incubated under normoxia (≈18% pO2) without treatment, or with either 250 µM DFX and 100 µM DMOG, respectively (scale indicated on the figures; n = 3).
Figure 5.
Reduction of HIF-1α of human MSCs (i) enhances adipogenesis under normoxic conditions, (ii) partially restores hypoxia-induced attenuation of adipogenesis and (iii) suppresses hypoxia-enhanced osteogenesis.
(a) Transduction of anti HIF-1α-shRNA-constructs efficiently reduced HIF-1α protein expression as shown by immunoblot (2-weeks data). (b) Oil-Red-O stain for the analysis of adipogenesis of shRNA-construct transduced MSCs and (c) von-Kossa stain for the analysis of osteogenesis of shRNA-construct transduced MSCs. Cells were maintained under normoxia (≈18% pO2) or hypoxia (1% pO2) for 4 weeks using either osteogenic or adipogenic differentiation medium (scale indicated on the figure; n = 3).
Table 4.
Patient information and experiments conducted.
Table 5.
Primersets used.