Direct contact is required for HSC survival/proliferation on AFT024 cocultures. (A) Schematic representation of the experimental procedure for the conditioned media exchange approach (top panel): irradiated AFT024 stroma was cultured on “Dexter-type” media for 2 or 4 days, before conditioned media were transferred to 2018 stroma (or vice versa). Percentage of dividing founder HSCs cultured on 2018 cells in the presence of AFT024 media conditioned for 2 days (n = 4 independent experiments, 168 trees) or 4 days (n = 3 independent experiments, 162 trees) are compared with the 2018 control (no media exchange, white bar). In addition, the respective percentage of dividing HSCs cultured on AFT024 stroma with 2018-conditioned media for 2 days (n = 3 independent experiments, 149 trees) and 4 days (n = 3 independent experiments, 175 trees) is compared with the AFT024 control (dark gray bar). (B) Schematic representation of the experimental procedure for continuous media conditioning (top panel): AFT024 stroma surrounding a physically separated (silicon insert) island of 2018 cells (or vice versa). Area covered by the surrounding stroma is ∼8 times larger. HSCs were exclusively cultured in contact with the inner stroma compartment, but exposed to media mainly conditioned by the outer stroma (∼6 times more cells). Generation-based analysis of dividing HSCs cultured on 2018 stroma while exposed to AFT024 conditioned media (n = 3 independent experiments, 194 trees) or vice versa (n = 3 independent experiments, 141 trees). White and dark gray bars represent control conditions. (C) Quantification of HSC divisional rates after culture on different ratios of AFT024 and 2018 stroma: 100% to 0% (AFT024 control, dark gray bar), 90% to 10% (n = 5 independent experiments, 180 trees), 50% to 50% (n = 4 independent experiments, 122 trees), 10% to 90% (n = 4 independent experiments, 120 trees), and 0% to 100% (2018 control, white bar) respectively. (D) Snapshots from time-lapse imaging experiment showing the different channels acquired. Stroma cells were differentially transduced with lentiviral vectors expressing distinct fluorescent proteins fused with the c-HA-Ras farnesylation signal domain for membrane anchoring allowing visualization of the entire cell volume (including cell protrusions). (E) Bar chart representing the percentage of cell lifetime, for which dividing (left panel) or dying HSCs (right panel) were adherent to AFT024 (black bar), 2018 (white bar), or both stroma (gray bar) (n = 3 independent experiments, 47 trees). (F) Violin plots depicting cell lifetime of dying founder HSCs cultured on AFT024 (n = 7 independent experiments, 49 trees), 2018 (n = 5 independent experiments, 184 trees), or 2012 stroma (n = 3 independent experiments, 75 trees). Black lines represent the median. Data were compared using the rank-based nonparametric Kruskal-Wallis test with Dunn post-hoc test. CM, conditioned media; mseCFP, monomeric super enhanced cyan fluorescent protein; ns, nonsignificant; tdTOMATO, tandem dimer Tomato.