Antagonism of PPAR-γ signaling expands human hematopoietic stem and progenitor cells by enhancing glycolysis

Abstract

Hematopoietic stem cells (HSCs) quiescently reside in bone marrow niches and have the capacity to self-renew or differentiate to form all of the blood cells throughout the lifespan of an animal. Allogeneic HSC transplantation is a life-saving treatment for malignant and nonmalignant disorders. HSCs isolated from umbilical cord blood (CB) are used for hematopoietic cell transplantation (HCT), but due to the limited numbers of HSCs in single units of umbilical CB, a number of methods have been proposed for ex vivo expansion of human HSCs. We show here that antagonism of peroxisome proliferator-activated receptor (PPAR)-γ promotes ex vivo expansion of phenotypically and functionally defined subsets of human CB HSCs and hematopoietic progenitor cells (HSPCs). PPAR-γ antagonism in CB HSPCs strongly downregulated expression of several differentiation-associated genes, as well as fructose-bisphosphatase 1 (FBP1; which encodes a negative regulator of glycolysis), and enhanced glycolysis without compromising mitochondrial metabolism. The expansion of CB HSPCs by PPAR-γ antagonism was completely suppressed by removal of glucose or inhibition of glycolysis. Moreover, knockdown of FBP1 expression promoted glycolysis and ex vivo expansion of long-term repopulating CB HSPCs, whereas overexpression of FBP1 suppressed the expansion of CB HSPCs that was induced by PPAR-γ antagonism. Our study suggests the possibility for a new and simple means for metabolic reprogramming of CB HSPCs to improve the efficacy of HCT.

Figure 1. PPARγ antagonism promotes ex vivo expansion of human CB HSPCs

(a) Left, the experimental strategy for the compound screen used to identify GW9662, a PPARγ antagonist, as promoting CB HSC ex vivo expansion. Freshly isolated CB CD34⁺ cells were cultured with vehicle control or compounds from the library for 4 days. Phenotypic HSC (pHSCs, Lin⁻CD34⁺CD38⁻CD45RA⁻CD49f⁺CD90⁺) expansion was determined by FACS analysis. Right, fold ex vivo expansion of CB pHSCs for the compounds tested. The red diamond indicates GW9662. (b) Quantification data of CB pHSC ex vivo expansion by vehicle or GW9662 at days 4 and 7. 50,000 CD34⁺ cells per well were plated at day 0 and the pHSC frequency was determined by flow cytometry. Data pooled from four independent experiments (n=12 cultures per group) are shown as box-and-whisker plots (the lines indicate median values, the whiskers indicate minimum and maximum values, the boxes indicate interquartile range). The exact cell numbers from each experiment are shown in Supplementary Table 1. One-way ANOVA; ***p<0.001. (c) Representative FACS plots showing expansion of CB pHSCs by GW9662. The pHSC population was assessed as Lin⁻CD34⁺CD38⁻CD45RA⁻CD49f⁺CD90⁺ cells in day 4 cultures. Gating was based on use of isotype control antibodies. The percentages indicate the frequencies of gated cell populations among the live cell events collected. Representative plots from 4 independent experiments are shown. (d) CFU numbers in 50,000 cells of day 0 uncultured CD34⁺ cells and the progeny of an equivalent number of CD34⁺ cells that were expanded in the presence of vehicle control or GW9662 for 4 days (n=6 cultures from two independent experiments per group). One-way ANOVA; **p<0.01, ***P<0.001.

Figure 2. PPARγ antagonism expands long term HSCs from human cord blood

(a) Representative FACS plots showing the percent engraftment of vehicle control- or GW9662-treated human CB CD34⁺ cells in the bone marrow of recipient NSG mice 4 months after transplantation. Non-transplanted mouse bone marrow cells were used as the negative control. Representative plots from 3 independent experiments are shown. (b) The percentage of human CD45⁺ cells in peripheral blood (PB) at 2 and 4 months, and the percentage of human CD45⁺ cells in bone marrow (BM) at 4 months, after transplantation of NSG mice with the progeny of 30,000 CB CD34⁺ cells treated with vehicle or GW9662 for 4 days (n=5 mice per group). Data are shown as dot plots (mean±s.e.m.). *p<0.05, **p=0.009. (c–e) Human CD33⁺ myeloid cell, CD3⁺ T cell and CD19⁺ B cell chimerism in the bone marrow (BM) of primary recipients at 4 months after transplantation (n=5 mice per group). Data are shown as dot plots (mean±s.e.m.). *p=0.032, **p<0.01. (f) The engraftment of human CD45⁺ cells in the peripheral blood and bone marrow of secondary recipient NSG mice at 4 months after transplantation (n=5 mice per group). Data are shown as dot plots (mean±s.e.m.). From (b) to (f), two-tailed Student’s t-test was used. *p=0.017, **p=0.002. (g,h) The frequency of human SRCs in uncultured CB CD34⁺ cells or in the progeny of an equivalent number of CD34⁺ cells treated by vehicle or GW9662 for 4 days. Graded doses of uncultured, vehicle or GW9662 treated CB CD34⁺ cells were transplanted into irradiated NSG mice and the percentage of human CD45⁺ cells in bone marrow (BM) was analyzed at 3 months after transplantation (n=2–5 mice per group, see Supplementary Table 2). Poisson statistical analysis of data from Supplementary Table 2 and 3 is shown in (g). The percentage of negative mice for each dose of cells is plotted. Inverted triangles indicate that all tested mice were positive in this group. Solid lines indicate the best-fit linear model for each data set. Group A (black line) corresponds to the uncultured group, group B (red line) corresponds to the vehicle-treated group, and group C (green line) corresponds to the GW9662-treated group. Dotted lines represent 95% confidence intervals. HSC frequencies (line in the box) and 95% confidence intervals (box) presented as the number of SRCs in 1 × 10⁶ CD34⁺ cells are shown in (h). NS, not significant; *p=0.0375, **p=0.00828. Results from another independent experiment are shown in Supplementary Figure 6b,c and Supplementary Tables 4 and 5.

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

(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.

Figure 4. Loss of function of FBP1 expands CB HSPCs by enhancing glycolysis

(a) Quantification of CB HSC ex vivo expansion by vehicle or MB05032 at days 4 and 7. 50,000 CD34⁺ cells per well were cultured and the phenotypic HSC frequency was determined at days 4 and 7. Data pooled from four independent experiments are shown as box-and-whisker plots (n=12 cultures per group). The exact cell numbers from each experiment are shown in Supplementary Table 6. One-way ANOVA; ***p<0.001. (b) Expansion of CB HSPCs transduced with scrambled control shRNA or FBP1 shRNA and cultured for 4 days, as determined by FACS. Data pooled from two independent experiments are shown (n=6 cultures per group). Two-tailed Student’s t-test was used. **p=0.0015. (c) Expansion of CB HSPCs transduced with control vector (pEGFP-N1) or pEGFP-N1-FBP1 and then cultured in expansion medium containing vehicle or GW9662 for 4 days, as determined by FACS (n=3 independent experiments). One-way ANOVA; NS, not significant; **p<0.01. (d,e) The frequency of human SRCs in control vector or FBP1 transfected CB CD34⁺ cells treated with vehicle or GW9662. Graded doses of GFP⁺CD34⁺ cells were transplanted into irradiated NSG mice. The percentage of human CD45⁺ cells in bone marrow (BM) was analyzed 2 months after transplantation (n=4–5 mice per group, see Supplementary Table 7). Poisson statistical analysis of data from Supplementary Table 7 is shown in (d). The percentage of negative mice for each dose of cells is plotted. Inverted triangles indicate that all tested mice were positive in this group. Solid lines indicate the best-fit linear model for each data set. Group A (black line) corresponds to the vehicle & vector group, and group B (red line) corresponds to the GW9662 & vector group, and group C (green line) corresponds to the vehicle & FBP1 group, group D (blue line) corresponds to the GW9662 & FBP1 group. Dotted lines represent 95% confidence intervals. HSC frequencies (line in the box) and 95% confidence intervals (box) presented as the number of SRCs in 1 × 10⁶ GFP⁺CD34⁺ cells are shown in (e). NS, not significant; *p=0.021, **p=0.001. (f) Human CD45⁺ cell chimerism, human CD33⁺ myeloid cell chimerism and human CD19⁺ B cell chimerism in bone marrow (BM) of NSG mice at 4 months after transplantation of the progeny of 10,000 CB CD34⁺ cells treated with vehicle or MB05032 for 4 days (n=5 mice per group). Another independent experiment is shown in Supplementary Fig. 9h–k. Data are shown as dot plots (mean±s.e.m.). Two-tailed Student’s t-test was used. *p<0.05, ***p<0.001. (g,h) The frequency of human SRCs in uncultured CB CD34⁺ cells, and the progeny of an equivalent number of CD34⁺ cells treated by vehicle or MB05032. Graded doses of uncultured, vehicle or MB05032 treated CB CD34⁺ cells were transplanted into irradiated NSG mice. The percentage of human CD45⁺ cells in bone marrow (BM) was analyzed 4 months after transplantation (n=4–5 mice per group, see Supplementary Table 4). Poisson statistical analysis of data from Supplementary Tables 4 and 5 is shown in (g). The percentage of negative mice for each dose of cells is plotted. Inverted triangles indicate that all tested mice were positive in this group. Solid lines indicate the best-fit linear model for each data set. Group A (black line) corresponds to the uncultured group, and group B (red line) corresponds to the vehicle-treated group, and group C (green line) corresponds to the MB05032-treated group. Dotted lines represent 95% confidence intervals. HSC frequencies (line in the box) and 95% confidence intervals (box) presented as the number of SRCs in 1 × 10⁶ CD34⁺ cells are shown in (h). NS, not significant; *p<0.05. Data from another independent experiment is shown in Supplementary Figure 10b,c and Supplementary Tables 8 and 9. (i) ECAR measurements in purified CB CD34⁺ cells following a 4-day culture with vehicle or MB05032. 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<0.01; ***p<0.001. (j) Relative glucose uptake in vehicle or MB05032 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”. Two-tailed Student’s t-test was used. ***p<0.001. (k) Relative levels of lactate secreted by vehicle or MB05032 treated CB CD34⁺ cells (n=3 independent experiments). Two-tailed Student’s t-test was used. ***p<0.001. (l) CB HSC ex vivo expansion by vehicle or MB05032 in complete medium (with glucose) and glucose free medium. 50,000 CD34⁺ cells per well were plated in the indicated culture medium containing 10% dialyzed serum, and the phenotypic HSC frequency was determined by flow cytometry at day 4. Data pooled from three independent experiments are shown as box-and-whisker plots (n=9 cultures per group). One-way ANOVA; ***p<0.001. (m) CB HSC expansion in the presence of vehicle, MB05032, 2-DG (1 mM) or MB05032+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 (n=6 cultures per group). For box-and-whisker plots in (a), (l) and (m), the lines indicate median values; the whiskers indicate minimum and maximum values; the boxes indicate interquartile range. One-way ANOVA; ***p<0.001.