Extended Data Figure 5. Ptpn11^E76K/+ MSPCs do not directly activate HSCs.

BM-derived MSPCs were enriched from Ptpn11^E76K/+Nestin-Cre⁺ and Ptpn11^+/+Nestin-Cre⁺ mice, as described in Methods. MSPCs at the 2nd or 3rd passages were plated in regular 24-well plates (a) or lower chambers of transwells (b). Forty-eight hours later when the cells were confluent, HSCs (75–200) (Lin⁻Sca-1⁺c-Kit⁺CD150⁺CD48⁻Flk2⁻) sorted from Ptpn11^E76K/+Mx1-Cre⁺ and Ptpn11^+/+Mx1-Cre⁺ mice (8 weeks after pI–pC administration) were seeded in the same wells (a) or in upper chambers with the 0.4 µm pore size (b). The cells were co-cultured in StemSpan medium supplemented with cytokines TPO (50 ng ml⁻¹), Flt3 ligand (50 ng ml⁻¹), SCF (50 ng ml⁻¹), IL-3 (20 ng ml⁻¹), and IL-6 (20 ng ml⁻¹). Frequencies of myeloid (Mac-1⁺Gr-1⁺) cells that differentiated from HSCs were assayed by FACS analyses after 7–10 days of co-culture. Experiments were performed three times and similar results were obtained in each (see Supplementary Information). Results shown are mean ±s.d. of triplicates from one experiment; N.S., not significant. Source data are available online.