Volume 45, Issue 2, Pages 346-357 (August 2016) Cellular Barcoding Links B-1a B Cell Potential to a Fetal Hematopoietic Stem Cell State at the Single-Cell Level  Trine A. Kristiansen, Elin Jaensson Gyllenbäck, Alya Zriwil, Tomas Björklund, Jeremy A. Daniel, Ewa Sitnicka, Shamit Soneji, David Bryder, Joan Yuan  Immunity  Volume 45, Issue 2, Pages 346-357 (August 2016) DOI: 10.1016/j.immuni.2016.07.014 Copyright © 2016 Elsevier Inc. Terms and Conditions

Immunity 2016 45, 346-357DOI: (10.1016/j.immuni.2016.07.014) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 1 Experimental Design for Cellular Barcoding of FL HSPCs (A) Schematic of the hematopoietic hierarchy illustrating heterogeneity within the LSK compartment. (B) FACS plots for LSK and HSC gating. (C) HSC (LSK CD150+CD48−) frequency within the E14.5 FL LSK compartment. Data were compiled from two litters (6+3 individual embryos) in two separate experiments, mean ≈ 5.9%. Error bars represent standard deviation of the mean. (D) Summary of experimental workflow. 104 sorted LSK cells were transduced at 15%–30% efficiency with LV-GFP-lib and transplanted into each lethally irradiated recipient together with 2 × 105 total BM support cells. After 16–20 weeks, peritoneal cavity B-1a cells, splenic follicular B-2 cells, and splenic granulocytes (Gr) were FACS sorted (as shown in Figure S1C). The DNA barcodes from each population were amplified in two parallel PCR reactions and sequenced in individual reactions (Seq A and B). Log transformed frequencies of filtered reads from the two technical replicates were plotted and Pearson correlation coefficients of r > 0.85 were accepted. Only barcodes present in both technical replicates were considered for further analysis (Experimental Procedures). Immunity 2016 45, 346-357DOI: (10.1016/j.immuni.2016.07.014) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 2 A Shared FL Progenitor of B-1a and B-2 Cells Is Revealed by Cellular Barcoding (A) Venn diagram representing the qualitative overlap between barcodes retrieved from peritoneal cavity B-1a (red), splenic follicular B-2 (blue), and splenic granulocytes (yellow) 16–20 weeks after FL E14.5 LSK transplantation. Filtered barcodes from each population were qualitatively compared to identify sequences represented in multiple populations. Linked comparison to T and erythroid lineage retrieved barcodes is shown in Figure S2A. Results are from a single recipient mouse and representative of three biological replicates in three individual experiments (Figure S2C). (B–J) Read frequency analyses for the indicated populations: Stacked bar graphs (B, E, H) illustrate the distribution of barcode read frequencies; pie charts (C, F, I) show the contribution of the indicated founder groups; and bar graphs (D, G, J) show the average read frequency of individual barcodes within each founder group. Error bars indicate standard error of the mean across individual barcodes. Immunity 2016 45, 346-357DOI: (10.1016/j.immuni.2016.07.014) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 3 A Shared FL Progenitor of B-1a and B-2 Cells Is Revealed by Single FL HSC Transplantations A total of 40 lethally irradiated recipients were transplanted with single E14.5 FL HSCs (LSK CD150+CD48–FLT3–) in two separate experiments. (A) Schematic of experimental setup detailing congenic backgrounds of donors and recipients used. Different congenic backgrounds were used in the two separate experiments to control for potential bias. (B) 3 weeks after transplantation, donor cell contribution to peripheral blood mononuclear cells (PBMCs) was assessed by FACS analysis. (C) Peritoneal cavity analyses of recipients were performed at 7 weeks after transplantation. FACS analysis of peritoneal cavity B-1a (red gates) and B-2 (blue gates) are shown. Immunity 2016 45, 346-357DOI: (10.1016/j.immuni.2016.07.014) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 4 Serial Transplantations of Barcoded LSK Cells Demonstrate that Initially B-1a Potent FL HSCs Become B-2 Restricted with Age (A) Experimental design for secondary transplantations of barcoded FL LSKs. LSKs were sorted from the primary recipient shown in Figure 2A and transplanted into two recipients to allow for maximum recovery of serially transplantable founder clones. (B) 27 barcodes were retrieved from the B-2 population of the two secondary recipients combined (black dots). Their original lineage contribution within the primary recipient is shown by superimposing the 27 barcodes onto the appropriate output patterns of the Venn diagram from Figure 2A. The associated barcode sequences were used to link the clonal behavior from the primary to secondary recipients and are listed to the right. (C) FACS analysis of peritoneal cavity GFP+CD19+ B cells in the primary and secondary recipients of barcoded FL LSK cells at 16 weeks after transplantation shows diminished B-1a potential at the time of secondary transplantation. Immunity 2016 45, 346-357DOI: (10.1016/j.immuni.2016.07.014) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 5 Loss of B-1a Potential Correlates with HSC Intrinsic Molecular Changes during Unperturbed Development (A) Cellular barcoding experiments of LSK cells from E14.5 FL, 1- to 5-day-old newborn (NB) BM, and 3-month-old adult BM (ABM). Granulocyte contributing barcodes representing founder HSCs are highlighted. Venn diagrams show qualitative barcode overlap between B-1a (red), B-2 (blue), and granulocytes (Gr, yellow) at 16–20 weeks after transplantation. The fraction of B-1a/B-2/Gr founders among traced HSCs display a developmental decline. Each Venn diagram is representative of 1–3 biological and technical replicates (Figures S2C and S2D). (B and C) Gene expression analysis using the Biomark platform. Results include four biological replicates for each developmental time point in two separate experiments. (B) Single-cell gene expression of HSCs from E14.5 FL (46 cells), 19-day-old BM (46 cells), and 3-month-old ABM (70 cells). Single HSCs (LSK CD150+CD48–FLT3–) were sorted and assessed for the presence of the indicated transcripts. Each column represents a single HSC. Expression data for each gene is displayed as relative Ct values across all cells assessed. (C) Quantitative gene expression analysis was performed on bulk HSCs (ten cells) in parallel with single-cell analysis. Dot plots display mean relative gene expression ± standard error of the mean as normalized to Hprt. Each dot represents the average of the two technical replicate wells. p values were derived from Kruskal-Wallis test with Dunns multiple comparisons test. ∗p < 0.05; ∗∗p < 0.005. Immunity 2016 45, 346-357DOI: (10.1016/j.immuni.2016.07.014) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 6 Lin28b Confers a Fetal-like HSC Behavior Barcoded LSK cells from untreated tet-Lin28b or littermate control (CTRL) adult BM were transplanted into doxycycline-treated recipient mice to induce constitutive transgene expression (Figure S5A). (A and B) Barcode analysis of tet-Lin28b and CTRL recipients at 16–20 weeks after transplantation. Venn diagrams illustrate the qualitative barcode overlap between peritoneal cavity B-1a (red), splenic follicular B-2 (blue), and splenic granulocyte (Gr, yellow). Data are representative of two separate biological and experimental replicates (Figure S2E). (C–E) Read frequency analyses for the indicated populations: Stacked bar graphs illustrate the distribution of barcode read frequencies; pie charts show the contribution of the indicated founder groups; and bar graphs show the average read frequency of individual barcodes within each founder group. Error bars indicate standard error of the mean across individual barcodes. (F) Representative FACS analysis of donor-derived lineage-negative BM cells from tet-Lin28b and CTRL recipients 16–20 weeks after transplantation. (G) Quantification of CD150+CD48– HSC frequency among lineage-depleted donor cells. Five tet-Lin28b and three CTRL recipients were analyzed in two separate experiments. p value was derived from the Mann-Whitney U test. Error bars represent standard deviation of the mean. (H) The read numbers of barcodes retrieved from both the LSK (y axis) and Gr (x axis) population of one tet-Lin28b recipient were plotted against each other. Pearson correlation coefficient is indicated (r). The eight most frequent barcodes from both populations were also B-1a potent (dotted circle). (I) Schematic model for multi-parametric analyses of upstream HSC abundance and downstream mature lineage representation, clonally linked through unique barcode identities. Immunity 2016 45, 346-357DOI: (10.1016/j.immuni.2016.07.014) Copyright © 2016 Elsevier Inc. Terms and Conditions