Normal HSCs are aberrantly activated by Ptpn11^E76K/+ neoplastic cells, leading to accelerated differentiation and exhaustion. (A) Genomic DNA was extracted from LSK cells (Lin⁻Sca-1⁺c-Kit⁺), myeloid cells (Mac-1⁺Gr-1⁺), monocytes (CD115⁺Gr-1⁺), and B cells (B220⁺) isolated from the BM of Ptpn11^E76K/+LysM-Cre⁺ mice and Ptpn11^+/+LysM-Cre⁺ littermates. The abundance of the inhibitory neo cassette with a stop codon in the targeted Ptpn11 allele was determined by quantitative polymerase chain reaction (n = 5 mice per genotype). (B) BM cells harvested from 5- to 6-month-old Ptpn11^E76K/+LysM-Cre⁺ mice and Ptpn11^+/+LysM-Cre⁺ littermates were assayed for the frequencies of common myeloid progenitors (CMPs), granulocyte macrophage progenitors (GMPs), megakaryocyte erythroid progenitors (MEPs), and common lymphoid progenitors (CLPs; n = 6 mice per genotype). (C) BM cells (2 × 10⁴ cells) collected from 5- to 6-month-old Ptpn11^E76K/+LysM-Cre⁺ mice and Ptpn11^+/+LysM-Cre⁺ littermates (n = 3 mice per genotype) were processed for colony-forming unit assays. (D-E) Frequencies of HSC-enriched LSK (Lin⁻Sca-1⁺c-Kit⁺) cells (D) and HSCs (Lin⁻Sca-1⁺c-Kit⁺CD150⁺CD48⁻Flk2⁻) (E) in the BM and spleens of 5- to 6-month-old Ptpn11^E76K/+LysM-Cre⁺ mice and Ptpn11^+/+LysM-Cre⁺ littermates (n = 6 mice per genotype) were determined by multiparameter fluorescence-activated cell sorting (FACS) analyses. (F-H) BM cells freshly isolated from Ptpn11^E76K/+LysM-Cre⁺ mice and Ptpn11^+/+LysM-Cre⁺ littermates were assayed by FACS analyses to determine apoptotic cells (n = 6 mice per genotype) (F), cell cycle distribution (n = 5 mice per genotype) (G), and levels of phosphorylated Erk (p-Erk), p-Akt, and p-NF-κB in HSCs (n = 4 mice per genotype) (H). (I-J) Bone sections prepared from 4- to 6-month-old Ptpn11^E76K/+LysM-Cre⁺ mice and Ptpn11^+/+LysM-Cre⁺ littermates were processed for immunofluorescence staining with the indicated antibodies. Spatial relationship between HSCs (Lin⁻CD48⁻CD41⁻CD150⁺) and endothelial (CD31⁺CD144⁺) cells was examined (representative images from n = 5 mice per genotype are shown); the distance of these 2 types of cells was calculated (I). Spatial relationship between HSCs (Lin⁻CD48⁻CD41⁻CD150⁺) and mesenchymal stem progenitor cells (MSPCs; Nestin⁺) was examined (representative images from n = 6 mice per genotype are shown); HSCs within 8 μm of MSPCs were considered to be close to MSPCs (J). (K-N) BM cells harvested from 3-month-old Ptpn11^E76K/+LysM-Cre⁺ mice (CD45.2⁺RFP⁻) and WT RPF transgenic mice (CD45.2⁺RFP⁺) were mixed at the HSC ratio of 1:1. The mixed BM cells and BM cells isolated from WT RPF transgenic mice (CD45.2⁺RFP⁺) were transplanted IV into lethally irradiated WT BoyJ mice (CD45.1⁺; n = 8 and 6 mice for mixed BM cells and WT BM cells, respectively). Sixteen weeks after transplantation, recipient mice were euthanized. Spleen weights (normalized against body weights) were documented (K). Frequencies of Mac-1⁺Gr-1⁺ cells in different donor-derived subpopulations in the peripheral blood (PB) were examined at the indicated time points (L). The pool sizes of HSCs (Lin⁻Sca-1⁺c-Kit⁺CD150⁺CD48⁻) (M) and the cell cycle distribution of HSCs (Lin⁻Sca-1⁺c-Kit⁺CD150⁺) in different donor-derived populations (N) were determined at 16 weeks after transplantation as above. *P = .05, **P = .01, ***P = .001. DAPI, 4′,6-diamidino-2-phenylindole; GM-CSF, granulocyte-macrophage colony-stimulating factor; ns, not significant.