Extended Data Figure 1: Optimizing conditions for long-term HSC culture.
(a) Schematic of the standard HSC culture assay: C57BL/6-CD45.1 BM CD34-cKit+Sca1+Lineage- (CD34-KSL) HSCs were sorted (50 cells/well) into U-bottom 96-well plate wells, see (b) for sorting scheme. HSC growth can be observed during culture by counting or flow cytometry, with media changes made every three days (after initial seven days in culture). After 7–28 days, functional HSC activity was determined using competitive transplantation into irradiated C57BL/6-CD45.2 mice, against 1×106 BM competitor cells from C57BL/6-CD45.1/CD45.2 (F1) mice. Donor chimerism within peripheral blood (PB) myeloid, T cell, and B cell lineages was determined after 4–16 weeks or longer. Where indicated, secondary transplantation assays were performed by transplanting 106 BM cells from primary recipients into irradiated C57BL/6-CD45.2 mice.
(b) FACS gating strategy for sorting CD34-KSL cells (gates 1–7) and CD150+CD34-KSL cells (gates 1–8) from cKit-enriched mouse BM. Representative of at least 5 experiments.
(c) Flow cytometric histograms for cell surface cKit staining of HSCs following stimulating with 100 ng/ml TPO and either 0, 10, or 100 ng/ml SCF for 1, 24, and 48 hours. Representative of 3 independent cultures.
(d) Mean florescence intensity (MFI) of cKit antibody staining on HSCs cultured in 100 ng/ml TPO supplemented with 10 ng/ml or 100 ng/ml SCF, analyzed after 1–72 hours in culture, relative to cultures containing 100 ng/ml TPO without SCF. Mean of 3 independent cultures. Error bars denote s.d.
(e) Mean 16-week donor PB chimerism from 1×104 HSC-derived cells following a 28-day culture on plastic (n=5), Collagen1 (n=3), Collagen4 (n=4), Fibronectin (n=3), Gelatin (n=5), or Laminin511 (n=4) culture plates (cultured in 100 ng/ml TPO and 10 ng/ml SCF with complete media changes). Competitive transplantation against 1×106 BM competitors.
(f) Number of live cells after culturing 50 CD34-KSL HSCs for 28 days on plastic (tissue culture-treated) plates or fibronectin-coated plates. Mean of 3 independent cultures. Error bars denote s.d.