Extended Data Fig. 2: Identification of PVA-based HSC culture conditions.

a, Fold change in MFI from cytokine immunoassays performed on HSA-based HSC cultures between day 8 and day 13. Medium changes performed at day 7 and day 10. Mean of four independent cultures with fold change relative to unconditioned medium. Error bars denote s.d. b, Mean expansion of 50 CD34⁺KSL haematopoietic progenitor cells at day 7, in 100 ng ml⁻¹ TPO and 10 ng ml⁻¹ SCF with or without addition of 0.3 ng ml⁻¹ to 10 ng ml⁻¹ mouse IL-6 (n = 4 cell cultures). c, Heat map displaying the MFI fold change from cytokine immunoassays using conditioned medium from HSC cultures at day 14. CD34⁻KSL HSCs were isolated from C57BL/6 wild-type (WT), Tlr2 knockout (TLR2-KO) or Tlr4 knockout (TLR4-KO) mice, and cultured in HSA-based cultures. Dexamethasone (+Dex) at 50 nM was added, where indicated. Mean of four independent cultures with fold change relative to unconditioned medium. d, Concentration of IL-6 observed in HSA-based cultures at day 14 of wild-type HSCs (n = 8 cell cultures), Tlr2 knockout HSCs (n = 8 cell cultures), Tlr4 knockout HSCs (n = 6 cell cultures), or wild-type HSCs + dexamethasone (n = 8 cell cultures). Error bars denote s.d. e, Mean donor peripheral-blood chimerism at week 12, from HSCs cultured for 7 days, in fresh medium (n = 7 mice) or in medium composed of 50% medium collected from a 12-day-long HSC culture and 50% fresh medium (termed ‘conditioned media’, n = 7 mice). Competitive transplantation against 1 × 10⁶ bone-marrow competitors. f, Example flow cytometry plots displaying c-KIT and SCA1 expression on the Lin⁻ progeny (left), and CD150 and CD48 expression in the KSL population (right) after a PVA-based HSC culture for seven days. Representative of four independent cultures. g, Concentration of various cytokines in conditioned medium at day 14, from HSA- or PVA-based CD34⁻KSL HSC cultures. Mean of eight independent cultures. Error bars denote s.d. Statistical significance was calculated using t-tests. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. h, Relative expression of p16Ink4a, p19Arf and Trp53 in KSL cells collected from cultures at day 14 (HSA-based cultures with half-medium changes, HSA-based cultures with complete medium changes and PVA-based cultures with complete medium changes), relative to expression in freshly isolated KSL cells. Mean of three independent cultures, with gene expression normalized to Gapdh expression. Error bars denote s.d. i, Number of phospho-γ-histone 2A.X (H2A.X) nuclear foci in KSL cells at day 28, from HSA-based or PVA-based HSC cultures. Irradiated cells were included as a positive control. Forty-nine cells quantified per condition. j, Relative expression of p16Ink4a, p19Arf and Trp53 in KSL cells collected from cultures at day 14 (left): HSA-based cultures, PVA-based cultures and PVA-based cultures supplemented with 1 ng ml⁻¹ lipopolysaccharide. Mean of technical quadruplets, with gene expression normalized to Gapdh expression. The concentration of IL-6 observed in these culture conditions is shown on the right. Mean of four independent cultures. Error bars denote s.d. k, Twenty-eight-day-long expansion of 50 CD150⁺CD34⁻KSL HSCs in medium containing 87% hydrolysed PVA or >99% hydrolysed PVA. Ten thousand cells at day 28 represent ~1 HSCeq for 87% PVA and ~5 HSCeq for 99% PVA. Mean of three independent cultures. Error bars denote s.d. l, Seven-day-long expansion of 50 human cord-blood CD34⁺ cells in HSA- or PVA-based cultures supplemented with 10 ng ml⁻¹ human SCF and 100 ng ml⁻¹ human TPO. Mean of three independent cultures. Error bars denote s.d.

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