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. Author manuscript; available in PMC: 2018 Mar 29.
Published in final edited form as: Nat Med. 2018 Jan 29;24(3):360–367. doi: 10.1038/nm.4477

Figure 3. PPARγ antagonism promotes ex vivo expansion of human CB HSCs by switching on FBP1-repressed glycolysis.

Figure 3

(a) Heat map showing gene expression downregulated by 4 days -GW9662 treatment of CB CD34+ cells as compared to vehicle treatment.

(b) Relative mRNA level of targeted genes downregulated by GW9662 was determined by quantitative real-time PCR (n=6 replicates from two independent experiments). Two-tailed Student’s t-test was used. **p<0.01, ***p<0.001.

(c,d) Western blot analysis of FBP1 expression in vehicle or GW9662 treated human CB CD34+ cells. Representative blot and quantification data from three independent experiments (n=3 independent experiments) are shown in (c) and (d). Actin was used as a loading control. Two-tailed Student’s t-test was used. **p=0.004. The uncropped blot was shown in Supplementary Fig. 13.

(e,f) FBP1 expression in vehicle or GW9662 treated human CB HSCs, as analyzed by FACS. Representative histogram and quantification data from three independent experiments (n=3 independent experiments) are shown in (e) and (f). Two-tailed Student’s t-test was used. ***p=0.0002.

(g) ECAR measurements in purified CB CD34+ cells following a 4-day culture with vehicle or GW9662. Data pooled from three independent experiments are shown as mean±s.d. (n=3 independent experiments). Two-tailed Student’s t-test was used. **p=0.0018, ***p=0.0008.

(h) Relative glucose uptake in vehicle or GW9662 treated CB CD34+ cells. Glucose uptake was determined by FACS based on the mean fluorescence intensity (MFI) of the incorporated fluorescent glucose analog 2-NBDG (n=3 independent experiments). The value of vehicle group was set as “1”. ***p=0.00016.

(i) Relative levels of lactate secreted by vehicle or GW9662 treated CB CD34+ cells (n=3 independent experiments). **p=0.0049.

(j) OCR measurements in purified CB CD34+ cells following a 4-day culture with vehicle or GW9662. Representative data from three independent experiments is shown as mean±s.d..

(k) CB HSC ex vivo expansion by vehicle or GW9662 in complete medium (with glucose) and glucose free medium. 50,000 CD34+ cells per well were cultured in the indicated culture medium containing 10% dialyzed serum, and the number of pHSCs was determined at day 4. Data pooled from three independent experiments are shown as box-and-whisker plots (the lines indicate median values, the whiskers indicate minimum and maximum values, the boxes indicate interquartile range) (n=9 cultures per group). One-way ANOVA; ***p<0.001.

(l) CB HSC expansion in the presence of vehicle, GW9662, 2-DG (1 mM) or GW9662+2-DG (1 mM). 50,000 CD34+ cells per well were cultured in the indicated conditions, and the number of pHSCs was determined at day 4. Data pooled from two independent experiments are shown as box-and-whisker plots (the lines indicate median values, the whiskers indicate minimum and maximum values, the boxes indicate interquartile range) (n=6 cultures per group). One-way ANOVA; **p=0.002, ***p<0.001.