1. Volume 71, Issue 3, Pages 201-209 (February 2007)
Chronic metabolic acidosis alters osteoblast differentiation from human mesenchymal stem cells 
S. Disthabanchong, P. Radinahamed, W. Stitchantrakul, S. Hongeng, R. Rajatanavin 
Kidney International 
Volume 71, Issue 3, Pages (February 2007)
DOI: /sj.ki
Copyright © 2007 International Society of Nephrology Terms and Conditions

2. Figure 1
Flow cytometry analysis. Flow cytometry of bone marrow mononuclear cells before plating for (a) CD45, CD105, (b) CD34, CD105 and after the fourth passage for (c) CD14, CD45, (d) CD34, (e) HLA-DR, and (f) CD105.
Kidney International  , DOI: ( /sj.ki )
Copyright © 2007 International Society of Nephrology Terms and Conditions

3. Figure 2
Morphology of human MSCs. Phase-contrast microscopy demonstrates (a) the spindle-shaped morphology of MSCs grown in the absence of OM, (b) nodular aggregates of mineralization in the presence of OM for 28 days, and (c) adipocytes with lipid-rich vacuoles.
Kidney International  , DOI: ( /sj.ki )
Copyright © 2007 International Society of Nephrology Terms and Conditions

4. Figure 3
Cell cytotoxicity and proliferation assay. MSCs were cultured in the absence of OM and HCl (pH 7.4) and with increasing amount of HCl (pH 7.25, 7.15, and 7). (a) Cell cytotoxicity as evaluated by lactate dehydrogenase-cytotoxicity assay. (b) Cell proliferation by 3-(4,5-dimethyl-thiazolyl-2)-2,5-diphenyltetrazolium bromide assay. There was no significant difference in the cytotoxicity assay. Results represent an average of three experiments. aP<0.05 compared to day 0, bP<0.05 compared to pH 7.4, *P<0.05 compared to pH 7.25 and 7.15, and #P<0.05 compared to pH 7.25.
Kidney International  , DOI: ( /sj.ki )
Copyright © 2007 International Society of Nephrology Terms and Conditions

5. Figure 4
Time course of the effect of chronic metabolic acidosis on the expression of osteoblastic genes. MSCs were grown in the presence of OM without HCl (pH 7.4 –•–) and with HCl (pH ▴--). Negative controls were cultivated in the absence of OM (pH ○--, and pH ▵--). Expression levels of mRNA normalized by 18s rRNA are expressed as fold increase from day 0. (a, c, e, f, g, and i) represent data from donor 1. (b, d, and h) Represent data from donor 2. Results represent an average of 5–8 experiments except single experiment for negative controls. aP<0.05 compared to day 0 or day 14 for osteocalcin, bP<0.05 compared to pH 7.4.
Kidney International  , DOI: ( /sj.ki )
Copyright © 2007 International Society of Nephrology Terms and Conditions

6. Figure 5
The effect of chronic metabolic acidosis on the expression of osteoblastic proteins. (a) Runx2, (b) osterix, and (c) ATF4. MSCs were grown in the presence of OM without HCl (pH 7.4) and with HCl (pH 7.15). Representative Western blot data of each protein from three experiments are shown.
Kidney International  , DOI: ( /sj.ki )
Copyright © 2007 International Society of Nephrology Terms and Conditions

7. Figure 6
The amount of collagen staining and alkaline phosphatase enzyme activity. MSCs were grown in the presence of OM without HCl (pH 7.4) and with increasing amount of HCl (pH 7.25, 7.15, and 7). Negative controls were cultivated in the absence of OM (pH ○--, and pH ▵--). (a) Collagen staining and (b) alkaline phosphatase enzyme activity were determined and normalized by protein concentration. The collagen experiments were performed using MSCs from donor 2. Results represent an average of six experiments except single experiment for negative controls. aP<0.05 compared to day 0, bP<0.05 compared to pH 7.4, *P<0.05 compared to pH 7.25 and 7.15, and #P<0.05 compared to pH 7.25.
Kidney International  , DOI: ( /sj.ki )
Copyright © 2007 International Society of Nephrology Terms and Conditions

8. Figure 7
Dose–response of the effect of chronic metabolic acidosis on mRNA levels of osteoblastic genes. (a) Runx2, collagen, and osteocalcin and (b) osterix, ATF4, and alkaline phosphatase. MSCs were grown in the presence of OM without HCl (pH 7.4) and with increasing amount of HCl (pH 7.25, 7.15, and 7). Cells were harvested for RNA extraction at day 10 for runx2, type I collagen, and alkaline phosphatase, day 15 for osterix and ATF4 and day 28 for osteocalcin. Expression levels of mRNA normalized by 18s rRNA are expressed as fold change from pH 7.4. Results represent an average of 5–8 experiments. bP<0.05 compared to pH 7.4, *P<0.05 compared to pH 7.25 and 7.15, and #P<0.05 compared to pH 7.25.
Kidney International  , DOI: ( /sj.ki )
Copyright © 2007 International Society of Nephrology Terms and Conditions

9. Figure 8
Mineralization studies. MSCs were grown in the presence of OM without HCl (pH 7.4) and with increasing amount of HCl (pH 7.25, 7.15, and 7). (a) Von Kossa staining for mineral, (b) cuboidal-shaped osteoblasts without mineral deposit at day 28 in pH 7, (c) Sirius red staining for collagen and (d) the amount of calcium deposition normalized by protein concentration. Results represent an average of six experiments. aP<0.05 compared to day 7, bP<0.05 compared to pH 7.4, and #P<0.05 compared to pH 7.25.
Kidney International  , DOI: ( /sj.ki )
Copyright © 2007 International Society of Nephrology Terms and Conditions