Journal: EMBO Reports

Article Title: Pyruvate metabolism guides definitive lineage specification during hematopoietic emergence

doi: 10.15252/embr.202154384

Figure Lengend Snippet: A Pyruvate is transported into mitochondria via the mitochondrial pyruvate carrier (MPC, inhibitor: UK5099) and converted to acetyl‐coA by pyruvate dehydrogenase complex (PDH, inhibitor: 1‐AA). Pyruvate dehydrogenase kinases (PDKs, inhibitor: DCA) negatively regulate PDH activity. B–E FACS‐sorted HE cells were subcultured with or without UK5099 (10 µM) or DCA (3 mM). Subculture day 3 representative GPA/CD43 plots (B, D) and subculture day 6 representative CD45/CD43 plots (C, E) are shown (see Fig for corresponding bar graphs). F Ratio of CFU‐E to CFU‐G, M, GM colonies ± SEM relative to the control condition obtained from HE cells subcultured with the indicated compounds for 6 days, see Fig for percentages ( n = 5 biological replicates with 1–2 technical replicates each, paired t ‐test). G Fold change in the expression of HBE1 or HBG1‐2 transcripts ± SEM normalized to KLF1 in CFUs obtained from HE cells treated with UK5099 (10 µM) or DCA (3 mM) relative to non‐treated cells ( n = 3 biological replicates, paired t ‐test). H Percentages of CD45 + CD56 + ± SEM cells obtained following 35‐day co‐culture of 3‐day subcultured HE cells with OP9‐DL1 stroma. During the 3‐day subculture, HE cells were treated with the indicated compounds. ( n = 3 biological replicates, one‐way ANOVA test, see Fig for plots). I–K Pregnant mice were injected with UK5099 or DCA at E9.5 and fetal livers were analyzed at E14.5 by flow cytometry. FL, fetal liver. Levels of T and B cells (I) and LT‐HSCs (J) as percentages in fetal liver are shown for control ( n = 10 biological replicates), UK5099‐treated ( n = 14 biological replicates), and DCA‐treated ( n = 16 biological replicates) conditions (one‐way ANOVA test). (K) The ratio of BFU‐E to CFU‐GM colonies obtained from sorted LT‐HSCs is shown (see also data in Appendix Fig ) (one‐way ANOVA test). CFU, colony‐forming unit; BFU, burst‐forming unit; E, erythroid; M, macrophage; G, granulocyte. L–P HE cells co‐cultured with OP9‐DL1 stroma were treated with DCA for 3 days and transplanted into irradiated NSG mice. Bone marrow (BM) and thymi were harvested on week 12. (L) The percentages ± SEM of human CD4 + CD8 + double‐positive thymocytes in huCD45 + cells from the thymus are shown (Control, n = 6 biological replicates; DCA, n = 7 biological replicates; unpaired t ‐tests). The percentages ± SEM of human B cells (M), CLPs (N) from the BM at week 12, as well as myeloid cells from PB at week 8 (O) and myeloid cells from BM at week 12 (P) in huCD45 + cells are shown (Control, n = 6 biological replicates; DCA, n = 7 biological replicates; unpaired t ‐tests). PB, peripheral blood. Data information: ns, not significant, * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: For single‐cell subcultures, single HE cells were directly sorted with a BD FACSAriaIII onto OP9‐DL1 stroma in flat‐bottom 96‐well plates with 120 μl OP9 medium (OptiMEM medium with Glutamax (Invitrogen) with 10% FCS, 1% penicillin–streptomycin solution (Thermo Fisher Scientific) and 1% 2‐mercaptoethanol (Invitrogen)) with SCF, IL‐6, IL‐11, IGF1, and EPO, and with or without 10 μM UK or 3 mM DCA.

Techniques: Activity Assay, Control, Expressing, Co-Culture Assay, Injection, Flow Cytometry, Cell Culture, Irradiation

Journal: EMBO Reports

Article Title: Pyruvate metabolism guides definitive lineage specification during hematopoietic emergence

doi: 10.15252/embr.202154384

Figure Lengend Snippet: A FACS‐sorted HE, EHT, or HSC‐like cells were subcultured with or without UK5099 (10 µM). Subculture day 3 CD43 + /GPA + cell frequencies ± SEM relative to the controls for all populations are presented (HE, n = 5 biological replicates; EHT, n = 6 biological replicates; HSC‐like, n = 4 biological replicates; paired t ‐tests). B FACS‐sorted HE cells were subcultured with or without 1‐AA (4 mM). Subculture day 3 CD43 + GPA + cell frequencies ± SEM relative to the control are shown ( n = 4 biological replicates, paired t ‐test). C Fold change of expression of MPC1 and MPC2 ± SEM relative to HPRT1 in shRNA‐transduced cells compared to shScrambled (shScr) is shown ( n = 3 biological replicates, unpaired t ‐tests). Untr, untransduced. D HE cells were transduced with shScrambled (shScr), shMPC1, shMPC2, or both the day after the sort and day 3 CD43 + /GPA + cell frequencies ± SEM relative to shScr are presented ( n = 4 biological replicates; one‐way ANOVA test). Untr, untransduced. E FACS‐sorted HE cells were stained with CTV and fluorescence was assessed by flow cytometry for GPA + cells at day 3 of subculture with or without UK5099 (10 µM). Representative of n = 3. F, G FACS‐sorted HE, EHT, or HSC‐like cells were subcultured with or without UK5099 (10 µM). Subculture day 6 CD43 + (F) and CD43 + CD45 + (G) cell frequencies ± SEM relative to the control for all populations are presented (HE, n = 7 biological replicates; EHT, n = 7 biological replicates; HSC‐like, n = 4 biological replicates; paired t ‐tests). H FACS‐sorted HE cells were subcultured with or without 1‐AA (4 mM). Subculture day 6 CD43 + and CD43 + CD45 + cell frequencies ± SEM relative to the control are shown ( n = 3 biological replicates, paired t ‐test). I, J FACS‐sorted HE cells were subcultured for 3 days with or without UK5099 (10 µM). CTV for HE‐derived CD45 + cells (I) and HE‐derived HSC‐like cell frequencies ± SEM relative to the control ( n = 7 biological replicates, paired t ‐test) (J) are shown. K–N FACS‐sorted HE and EHT cells were subcultured with or without DCA (3 mM). Subculture day 3 (K, n = 3 biological replicates) and day 6 (L, HE, n = 5 biological replicates; EHT, n = 4 biological replicates) CD43 + GPA + cell frequencies ± SEM relative to the controls are shown (paired t ‐test). (M) CTV for HE‐derived GPA + cells at subculture day 3 is shown. Representative of n = 3. (N) Subculture day 6 CD43 + CD45 + cell frequencies ± SEM relative to the controls for both populations are shown (HE, n = 5 biological replicates; EHT, n = 4 biological replicates; paired t ‐tests). O Fold change of expression of PDK1 , PDK2 , PDK3 , and PDK4 ± SEM relative to HPRT1 in shRNA‐transduced cells compared to shScrambled (shScr) is shown ( n = 3 biological replicates, paired t ‐tests). P HE cells were transduced with shScrambled (shScr) or shPDK1, shPDK2, shPDK3, or shPDK4 the day after the sort and day 6 CD43 + /CD45 + cell frequencies ± SEM relative to shScr are presented ( n = 3 biological replicates; one‐way ANOVA tests). Q CTV for HE‐derived CD45 + cells are shown. Representative of n = 3. R HE‐derived HSC‐like cell frequencies ± SEM relative to the control ( n = 4 biological replicates, paired t ‐test) are shown. S EdU incorporation ± SEM into HE cells was assessed by flow cytometry after a 24‐h pulse at days 1 and 2 of subculture with or without UK5099 (10 µM) or DCA (3 mM) ( n = 3 biological replicates). T, U Percentages of CFU assay colony types ± SEM obtained from HE cells subcultured with the indicated compounds for 3 days (U) ( n = 3 biological replicates, two‐way ANOVA test) or 6 days (T) ( n = 5 biological replicates, two‐way ANOVA test). CFU, colony‐forming unit; E, erythroid; M, macrophage; G, granulocyte; GEMM, mixed. V EryD and EryP CFU‐Es obtained from HE cells subcultured with the indicated compounds for 3 days. Scale bars, 100 µm. W Fold change in the expression of HBA1‐2 transcripts ± SEM normalized to KLF1 in CFUs obtained from HE cells treated with UK5099 (10 µM) or DCA (3 mM) relative to non‐treated cells ( n = 3 biological replicates, paired t ‐test). X Plots showing percentages of CD45 + CD56 + cells obtained following 35‐day co‐culture of 3‐day subcultured HE cells with OP9‐DL1 stroma. During the 3‐day subculture, HE cells were treated with the indicated compounds. Data information: ns, not significant, * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Article Snippet: For single‐cell subcultures, single HE cells were directly sorted with a BD FACSAriaIII onto OP9‐DL1 stroma in flat‐bottom 96‐well plates with 120 μl OP9 medium (OptiMEM medium with Glutamax (Invitrogen) with 10% FCS, 1% penicillin–streptomycin solution (Thermo Fisher Scientific) and 1% 2‐mercaptoethanol (Invitrogen)) with SCF, IL‐6, IL‐11, IGF1, and EPO, and with or without 10 μM UK or 3 mM DCA.

Techniques: Control, Expressing, shRNA, Transduction, Staining, Fluorescence, Flow Cytometry, Derivative Assay, Colony-forming Unit Assay, Co-Culture Assay

Journal: EMBO Reports

Article Title: Pyruvate metabolism guides definitive lineage specification during hematopoietic emergence

doi: 10.15252/embr.202154384

Figure Lengend Snippet:

Article Snippet: For single‐cell subcultures, single HE cells were directly sorted with a BD FACSAriaIII onto OP9‐DL1 stroma in flat‐bottom 96‐well plates with 120 μl OP9 medium (OptiMEM medium with Glutamax (Invitrogen) with 10% FCS, 1% penicillin–streptomycin solution (Thermo Fisher Scientific) and 1% 2‐mercaptoethanol (Invitrogen)) with SCF, IL‐6, IL‐11, IGF1, and EPO, and with or without 10 μM UK or 3 mM DCA.

Techniques: Recombinant, Saline, Flow Cytometry, Gene Expression, RNA Sequencing, Software