Figure 1. High-output ME- and MB-biased clones occur after erythropoietin (EPO) exposure and transplantation of hematopoietic stem and progenitor cells (HSPCs).

(a) HSPCs were sorted from the bone marrow of donor mice, lentivirally barcoded, cultured ex vivo with or without 1000 ng/ml EPO for 16 hr, and transplanted into sublethally irradiated mice. At week 4 post-transplantation, the erythroid (E), myeloid (M), and B-cells (B) lineages were sorted from the spleen and processed for barcode analysis. (b) The percentage of donor-derived cells (CD45.1⁺) among the total spleen, myeloid cells (CD11b⁺) or B-cells (CD19⁺) in the spleen of control and EPO group. (c) To better assess chimerism in erythroid cells, mTdTomato/mGFP donor mice were used. The fraction of Tom⁺ cells among erythroid cells (Ter119⁺) in the spleen and blood in control and EPO group. (d) The number of barcodes retrieved in the indicated lineages at week 4 after transplantation in the control and EPO groups. (e) Triangle plots showing the relative abundance of barcodes (circles) in the E, M, and B lineage with respect to the summed output over the three lineages (size of the circles) for the control and EPO groups. (f) Tthe percentage of HSPCs classified by the indicated lineage bias using a 10% threshold for categorization. (g) Quantitative contribution of the classes as in (f) to each lineage. Shown are values from several animals (n = 8 EPO, n = 10 control in b, n = 3 EPO, n = 4 control in c , spleen, n = 4 EPO, n = 8 control in c , blood collected over five different experiments d–g, n = 5 for the control group and n = 2 for the EPO group collected over one experiment). For all bar graphs, mean and SD between mice are depicted. Statistical significance tested using Mann–Whitney U-test p=0,05 for (b, c). Statistical significance tested by permutation test for different subsets in (g) (see Table 1).

Figure 1—figure supplement 1. Gating strategies and hematopoietic stem and progenitor cell (HSPC) marker expression after lentiviral transduction and ex vivo culture with or without erythropoietin (EPO).

(a) HSPCs were gated as propidium iodide-negative single C-Kit⁺ Sca-1⁺ Flt3^- CD150⁺ cells of C-Kit⁺-enriched bone marrow cells. (b) Erythroblast cells were gated as Ter119⁺ CD44⁺ FSC^hi cells on Ter⁺ enriched cells. (c) Gating strategy for B-cells (CD19⁺ CD11b^-), dendritic cells (CD19^- CD11b^- CD11c⁺), and myeloid cells (CD119^- CD11c^- CD11b⁺) on Ter119^- live single-donor cells. (d) Gating for MkP (C-Kit⁺ Sca-1^- CD150⁺ CD41⁺) from C-Kit⁺-enriched bone marrow cells. (e) Sort gating for GFP⁺ erythroid, myeloid, B-, and dendritic cells, respectively, used for barcoding analysis. (f) Representative flow cytometry plots of sorted HSPC pool after 6 hr lentiviral transduction and 16 hr ex vivo incubation with or without EPO.

Figure 1—figure supplement 2. Correlations in barcoding profiles of spleen and bone.

Hematopoietic stem and progenitor cells (HSPCs) were sorted from the bone marrow of donor mice, lentivirally barcoded, cultured ex vivo for 16 hr, and transplanted into sublethally irradiated mice. At week 4 post-transplantation, erythroid (E), B-cells (B), and the myeloid lineage (M) cells monocytes, eosinophils, neutrophils, and macrophages were sorted from the spleen and from bone and processed for barcode analysis. The myeloid lineage was merged according to the percentage of total donor myeloid each subset contributed as in Figure 3—figure supplement 1b. (a) Heatmaps showing the output of individual barcodes (rows) in different samples (columns) as indicated. Data is normalized by cell subset, log transformed, and clustered by complete linkage using Euclidean distance. No output is represented in black. (b) The percentage of barcodes in spleen and bone detected in the respective other organ. The Spearman rank correlation of barcodes in bone and spleen was for the B-, M-, and E-lineage 0.81, 0.69, and 0.7, respectively. Shown are values from several animals (n = 3). For all bar graphs, mean and SD between mice are depicted. Statistical significance tested using Mann–Whitney U-test p=0.05 for (b).

Figure 1—figure supplement 3. Characterization of lineage biases after transplantation of erythropoietin (EPO)-exposed hematopoietic stem and progenitor cells (HSPCs).

(a) Triangle plots from Figure 1e color coded by mice. (b) Quantitative contribution of the classes to each lineage as in Figure 1 using different thresholds of 0, 5, 10, 15, and 20%. (c–f) Data for an additional experiment as in Figure 1. (c) Number of barcodes retrieved in the indicated lineages at week 4 after transplantation in the control and EPO groups. (d) Triangle plots showing the relative abundance of barcodes (circles) in the erythroid (E), myeloid (M), and B-cell (B) lineage with respect to the summed output over the three lineages (size of the circles) for the control and EPO groups. (e) Proportion of HSPCs classified in the indicated lineage bias category using a 10% classification threshold. (f) Quantitative contribution of the classes as in (f) to each lineage. Shown are values from several animals (a–c, n = 5 for the control group and n = 2 for the EPO group [collected over one experiment], d–f, n = 2 for the control group and n = 3 for the EPO group [collected over one experiment]). For all bar graphs, mean and SD between mice are depicted. Statistical significance tested using Mann–Whitney U-test p=0.05 for (c, e).

Figure 1—figure supplement 4. High-output ME- and MB-biased hematopoietic stem and progenitor cells (HSPCs) occur 6 weeks after transplantation of erythropoietin (EPO)-exposed HSPCs.

HSPCs were sorted from the bone marrow of donor mice, lentivirally barcoded, cultured ex vivo with or without 1000 ng/ml EPO for 16 hr, and transplanted into sublethally irradiated mice. At week 6 post-transplantation, the erythroid (E), myeloid (M), and B-cells (B) lineages were sorted from the spleen and processed for barcode analysis. Quantitative contribution of HSPCs classified by the indicated lineage bias using a 10% threshold for categorization to each lineage. Shown are values from several animals (n = 2 EPO, n = 4 control). Mean and SD between mice are depicted.