Extended Data Fig. 4: Analysis of human reconstitution and correlation of cell cycle and culture time with the engraftment of MLLT3-OE cells. | Nature

Extended Data Fig. 4: Analysis of human reconstitution and correlation of cell cycle and culture time with the engraftment of MLLT3-OE cells.

From: MLLT3 governs human haematopoietic stem-cell self-renewal and engraftment

Extended Data Fig. 4

a, Transplantation strategy with ex vivo expanded FL-HSPCs transduced with control or MLLT3-OE vector. On day 5, transduced CD34+GFP+ cells derived from 1,000 HSPCs were sorted and replated in SFEM and transplanted into NSG mice at day 15. Human haematopoietic reconstitution was assessed at 24 weeks. Bone marrow from engrafted mice was transplanted to secondary recipients. b, FACS analysis showing human haematopoietic reconstitution (hCD45) (Fig. 1h) and multilineage differentiation (myeloid CD66, CD14; B-lymphoid CD19; T-lymphoid CD3, CD4, CD8). c, d, Representative FACS plots (c) and quantification (d) of human haematopoietic reconstitution from peripheral blood of transplanted NSG mice (extended from Fig. 1h). e, Quantification of human haematopoietic reconstitution from mouse spleen. Number of mice showing multi-lineage reconstitution versus total transplanted mice is shown (n = 13 or 14 mice, mean and individual values from four independent experiments). f, FACS sorting strategy for cell-cycle analysis after 100 min in vivo BrdU pulse in haematopoietic cells derived from human FL-HSPCs (8,000 GPI80+ FL-HSPCs or their progeny day 15 of culture after transduction with control or MLLT3-OE vector) transplanted in NSG mice and sorted 14 weeks after transplantation. g, h, Representative FACS plots (g) and quantification (h) of cell-cycle distribution by BrdU and DNA content (7AAD) staining. G0/G1 (BrdU7AADlo), S (BrdU+7AADint) and G2/M (BrdU7AADhi) phases are quantified in HSPCs, CD66b+ myeloid and CD19+ B-lymphoid cells (mean, n = 2). i, j, Cell-cycle analysis in HSPCs before transplantation, after 2-week culture in SFEM with SR1 and UM171. All P values determined by two-sided t-test. k, Transplantation strategy assessing the effect of extended culture on the reconstitution ability of MLLT3-OE FL-HSPCs. FL-HSPCs transduced with control or MLLT3-OE vector were sorted on day 5, and CD34+GFP+ cells derived from 1,000 uncultured HSPCs were transplanted directly into NSG mice or cultured in SFEM for an additional 10 days before transplantation. Numbers show mice with multilineage human engraftment, transplanted before (day 5) and after (day 15) culture expansion. l, m, Human CD45+ cells (l) and human HSPCs (m) in mouse bone marrow are quantified (n = 4 independent experiments; number of mice is indicated). *P < 0.05, two-sided t-test. n, o, Representative FACS plots (n) and quantification (o) of human haematopoietic (CD45) reconstitution and multilineage differentiation in mouse bone marrow after 18 weeks of secondary transplantation (mean, two independent experiments). Secondary mice were transplanted with 2-week-old bone marrow from primary recipients (1/2 of femur of the primary transplanted mice injected per recipient).

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